Mammalian cells for producing a secreted protein

ABSTRACT

The invention relates to the field of cell culture technology. It concerns the knockdown, using RNA interference, or gene knockout, of activating transcription factor 6 beta (ATF6B), or the combination of ceramide synthase 2 (CERS2) and TBC1 domain family member 20 (TBC1D20) proteins, which play central roles in the cellular secretion pathway. This downregulation leads to improved secretion of biopharmaceutically relevant products produced in mammalian cells. The invention specifically relates to mammalian cells having enhanced secretion of a recombinant therapeutic protein compared to a control cell, a method of producing said mammalian cell, a method for the production of a recombinant secreted therapeutic protein and the use of said mammalian cell for increasing the yield of a recombinant secreted therapeutic protein.

TECHNICAL FIELD

The invention relates to the field of cell culture technology. Itconcerns the knockdown, such as by using RNA interference, or geneknockout, of activating transcription factor 6 beta (ATF6B), or thecombination of ceramide synthase 2 (CERS2) and TBC1 domain family member20 (TBC1D20), proteins which play central roles in the cellularsecretion pathway. This downregulation leads to improved secretion ofbiopharmaceutically relevant products produced in mammalian cells. Theinvention specifically relates to mammalian cells having enhancedsecretion of a recombinant therapeutic protein compared to a controlcell, a method of producing said mammalian cell, a method for theproduction of a recombinant secreted therapeutic protein and the use ofsaid mammalian cell for increasing the yield of a recombinant secretedtherapeutic protein.

BACKGROUND

Improving titers of therapeutic proteins in production, making processesmore efficient, is a clear goal in industry. This can lead to reducedcosts and shortened timelines to supply protein material for clinicalstudies and markets. As overall yields in production processes aredetermined by cell specific productivity of the individual cell, as wellas the number of viable cells present in the process, strategies toimprove production efficiency usually aim to increase either of thesetwo parameters, without negatively affecting the other.

There is a need for improving recombinant protein production inmammalian cells, by increasing the specific productivity and/or thetotal yield (i.e., titer or concentration) of the protein in the cellculture supernatant, which is generally applicable and not dependent onthe individual cell line or protein to be produced.

Engineering strategies aiming to improve titers of therapeutic proteinscan focus on different stages of protein secretion, such as proteinfolding, post-transcriptional regulation and/or secretion. However, oneof the challenges associated with modifying the behaviour of cells toachieve favourable phenotypes for the production of recombinant proteinsis the complex nature of intracellular regulation circuits. Targetingthe expression of one gene or protein is not useful unless it is arate-limiting factor in a critical pathway or it is a transcriptionfactor with the potential to alter expression of a whole set of targetgenes. For this reason, microRNAs, as regulators of whole networks ofgenes, were used in protein-producing cell lines (WO 2013/182553).Targeting whole networks of genes is an undefined process unless alltarget genes are identified. Also regulation of some of those genes maynot be favourable for protein secretion or counteract the regulation ofother genes. It is an objective of the present invention to downregulatespecific proteins that constitute a bottleneck in protein secretion inorder to have a higher yield of secreted therapeutic proteins.

Expression of recombinant therapeutic proteins leads to an increase ofthe total protein amount that is translocated to the endoplasmicreticulum (ER) for further processing and subsequent secretion.Consequently, the protein folding capacity of the ER is one potentialbottleneck for protein production.

To this end, several studies are concerned with alterations in theunfolded protein response (UPR), an adaptive stress-response pathwaythat uses ER chaperones. Improved secretion of therapeutic proteins inrecombinant producer cells was obtained by overexpression of UPR relatedtranscription factors such as activating transcription factor 4 (ATF4)(Ohya T, et al., 2008. Biotechnology and Bioengineering. 100(2):317-324)the X-box binding protein-1 (XBP-1) (Tigges M, et al., 2006. MetabolicEngineering. 8:264-272; Becker E, et al., 2008. Journal ofBiotechnology. 135:217-223) or the CCAAT-enhancer-binding proteinhomologous protein (CHOP) (Nishimiya D, et al., 2013. Appl MicrobiolBiotechnol. 97:2531-2539). In 2009, Bommiasamy et al. showed that notonly overexpression of XBP-1, but also the expression of aconstitutively active form of activating transcription factor 6 alpha(ATF6a), increased the protein folding capacity of the ER (Bommiasamy etal., 2009, Journal of Cell Sciences. 122: 1626-1636).

A further bottleneck in the secretion of recombinant proteins is thedelivery from the ER to the Golgi apparatus. The small GTPase Rab1 has acrucial role in the vesicular transport of proteins that have beenprocessed in the ER. Due to its involvement in regulating the exit ofsecretory cargo from the ER and in COPII vesicle tethering (Haas A, etal., 2007. Journal of Cell Science. 120:2997-3010), an increase in Rab1activity could be advantageous for the overall protein secretion of thecell. While overexpression of Rab1 has been described to enlarge theGolgi apparatus and led to the up-regulation of vesicle traffic genesparticipating in the ER-to-Golgi transport, its inactivation ordepletion resulted in a blockage of ER exit (Haas A, et al., 2007.Journal of Cell Science. 120:2997-3010; Romero N, et al., 2013. MBoC.24:617-632). Rab1 is inactivated by a specific GTPase-activating protein(GAP), namely TBC1 Domain Family, Member 20 (TBC1D20) (Haas A, et al.,2007. Journal of Cell Science. 120:2997-3010).

In addition to limitations in the protein folding capacity of the ER andthe ER-to-Golgi transport, it has previously been shown that the proteintransport at the Golgi complex may be a further bottle neck inrecombinant protein secretion (Florin L, et al., 2009. Journal ofBiotechnology. 141:84-90). Ceramides are a source of diacylglycerol(DAG), which regulates the activity of protein kinase D (PKD) (HausserA, et al., 2005. Nat Cell Biol. 7(9):880-886; Fuchs Y F, et al., 2009.Traffic. 10:858-867; Baron C L and Malhotra V. 2002, Science 295(5553):325-8). PKD is crucial for the transport of proteins from the Golgi tothe plasma membrane by ceramide transfer protein (CERT) (Hanada K, etal., 2003. Nature. 426:803-809; Florin L, et al., 2009. Journal ofBiotechnology. 141:84-90; Fugmann T, et al., Journal of Cell Biology.178: 15-22). Ceramides are synthesized by six different isoforms ofCeramide Synthases (CERS1-6).

However, no specific proteins involved in the UPR or the secretorypathway have been identified that would loosen the above describedbottlenecks upon downregulation and hence result in increased productionof secreted proteins. Downregulation of genes may have technicaladvantages. For example, knock-out cells are stable with regard to thisphenotype and marker genes can be excised. Also, RNAi expression is moreeasily controlled since a saturating level is usually reached and theexpression level is less critical as for protein overexpression.

SUMMARY OF THE INVENTION

In the present invention it is shown that knocking-down selected targetgenes increased production of secreted proteins, particularly reducingATF6B activity, or reducing CERS2 and TBC1D20 activity, exerts apositive effect on secreted product concentration, protein productivityand viable cell density. These results suggest that the targeting ofATF6B, or the combination of CERS2 and TBC1D20, are effective strategiesfor improving titers of therapeutic proteins, including antibodies.

By virtue of downregulation of ATF6B, CERS2 and TBC1D20 proteinactivity, it is now possible to engineer mammalian cells to improvetheir cell productivity and/or cell viability, in particular, toincrease the amount of protein secreted by the cell culture. Themammalian cells, methods and uses provided herein allow for a moreefficient and cost-effective production of secreted proteins, especiallyfor antibody production. This may speed up drug development, since thegeneration of sufficient amounts of material for pre-clinical studies iscritical with regard to overall development timelines. The objects ofthe present invention can further be used for the generation of one orseveral specific secreted proteins for either diagnostic purposes,research purposes (target identification, lead identification, and leadoptimization) or manufacturing therapeutic proteins either on the marketor in clinical development.

In one aspect, the invention relates to a mammalian cell having enhancedsecretion of a recombinant therapeutic protein comprising (a) reducedexpression of the host cell proteins TBC1 domain family member 20(TBC1D20) and ceramide synthase 2 (CERS2); or (b) reduced expression ofthe host cell protein activating transcription factor 6 beta (ATF6B);wherein the mammalian cell further comprises one or more expressioncassette(s) encoding a recombinant secreted therapeutic protein.

Another aspect of the invention relates to a mammalian cell havingenhanced secretion of a recombinant therapeutic protein comprisingreduced expression of the host cell proteins TBC1D20 and CERS2.Optionally the mammalian cell may further comprise one or moreexpression cassette(s) encoding a recombinant secreted therapeuticprotein.

In the mammalian cell of the invention (a) the gene encoding the hostcell protein comprises a genetic modification that inhibits expressionof said host cell protein, or (b) the mammalian cell comprises a RNAoligonucleotide that inhibits expression of the gene encoding said hostcell protein by RNA-interference, wherein the protein expression ofTBC1D20 and CERS2 or the protein expression of ATF6B in the mammaliancell is reduced compared to the same mammalian cell not containing saidgenetic modification(s) or RNA oligonucleotide(s).

In a further aspect, the invention relates to a method of producing amammalian cell with enhanced secretion of a recombinant therapeuticprotein comprising (a) reducing expression of the host cell proteinsTBC1D20 and CERS2, or of the host cell protein ATF6B in the mammaliancell by introducing (i) a genetic modification into a gene encoding thehost cell protein that inhibits expression of said host cell protein, or(ii) a RNA oligonucleotide into the mammalian cell that inhibitsexpression of the gene encoding said host cell protein byRNA-interference, and (b) introducing one or more gene(s) encoding arecombinant secreted therapeutic protein. The method of the inventionmay further comprise the following steps: (c) selecting cells withenhanced secretion of the recombinant therapeutic protein; and (d)optionally culturing the cells obtained in step (c) under conditionswhich allow expression of one or more gene(s) encoding a recombinantsecreted therapeutic protein. According to the method of the inventionthe protein expression of TBC1D20 and CERS2 or the protein expression ofATF6B in the mammalian cell is reduced compared to the same mammaliancell not containing said genetic modification(s) or RNAoligonucleotide(s).

The RNA-interference in the cell of the invention or in the method ofthe invention may be mediated by small hairpin RNA (shRNA) or shortinterfering RNA (siRNA). Preferably the mammalian cell is transfectedwith one or more expression vector(s) comprising a nucleotide sequenceencoding said siRNA(s) or shRNA(s), more preferably the mammalian cellis stably transfected with one or more expression vector(s) encodingsaid siRNA(s) or shRNA(s). The siRNA used in the cell or the method ofthe invention may be (a) siTbc1D20 #1 (SEQ ID NO: 7) or siCerS2 #1 (SEQID NO: 8), or a combination thereof if TBC1D20 and/or CERS2 aretargeted; or (b) one or more of siAtf6b #1 (SEQ ID NO: 9), siAtf6b #2(SEQ ID NO: 10), and siAtf6b #3 (SEQ ID NO: 11) if ATF6b is targeted.The shRNA may comprise (a) shTbc1D20 #1 (SEQ ID NO: 12), or one or moreof shCerS2 #1 (SEQ ID NO: 13) and shCerS2 #2 (SEQ ID NO: 14), or acombination thereof, if TBC1D20 and/or CERS2 are targeted; or (b) one ormore of shAtf6b #1 (SEQ ID NO: 15) and shAtf6b #2 (SEQ ID NO: 37), ifATF6b is targeted. The exemplary siRNAs and shRNAs are particularlysuitable for hamster cells, such as CHO cells.

The genetic modification in the gene(s) encoding the host cellprotein(s) TBC1D20 and CERS2, or ATF6B may be independently (a) a genedeletion; or (b) a mutation in the gene that inhibits expression of thehost cell protein, preferably in the coding region of the gene and/orthe promoter or regulatory region of the gene. The host cell proteinTBC1D20 preferably has a sequence identity of at least 80% to the aminoacid sequence of SEQ ID NO: 4, the host cell protein CERS2 preferablyhas a sequence identity of at least 80% to the amino acid sequence ofSEQ ID NO: 5; and the host cell protein ATF6B preferably has sequenceidentity of at least 80% to the amino acid sequence of SEQ ID NO: 6.

According to the invention the recombinant secreted therapeutic proteinmay be an antibody, preferably a monoclonal antibody, a bi-specificantibody or a fragment thereof. Alternatively the recombinant secretedtherapeutic protein may also be a Fc-fusion protein. The cell accordingto the invention or used in the method of the invention is preferably arodent or a human cell, preferably a rodent cell, such as a CHO cell.

In a further aspect the invention also relates to a method for theproduction of a recombinant secreted therapeutic protein in a mammaliancell comprising (a) providing the mammalian cell of the invention,wherein the cell is transfected with a recombinant secreted therapeuticprotein or providing the mammalian cell produced by the method of theinvention, (b) culturing the mammalian cell of step (a) in a cellculture medium at conditions allowing production of the recombinantsecreted therapeutic protein, (c) harvesting the recombinant secretedtherapeutic protein, and optionally (d) purifying the recombinantsecreted therapeutic protein.

In yet another aspect, the invention relates to the use of the mammaliancell of the invention or to the mammalian cell produced by the method ofthe invention for increasing the yield of a recombinant secretedtherapeutic protein.

DESCRIPTION OF THE FIGURES

FIG. 1 shows IDENTIFICATION OF DIFFERENTIALLY EXPRESSED TARGET GENES OFMICRORNAS BY NEXT GENERATION SEQUENCING

Panel (A) is a table showing that CHO-DG44 cells stably secreting anIgG1 antibody (mAb1) (CHO-mAb1 cells) were transfected with each of thetwo microRNAs miR-1287 and miR-1978 having strong effects on antibodyproduction and specific productivity. 12 hours after transfectiontranscriptome profiling by next generation sequencing (NGS) was used toidentify direct target genes of the two miRNAs. Genes that weresignificantly downregulated with a |log 2| fold change>1 (geneexpression in untransfected control cells (mock) was more than 2-fold)were defined as hits. The miR-1287 target gene ATF6B and the miR-1978target genes CerS2 and Tbc1D20 were chosen for further analysis. Shownis the normalized fold change in expression (mock vs miRNA transfected)of the respective genes and its |log 2|. Effects of selected targetgenes on antibody production and specific productivity were firstassessed by siRNA-mediated gene knockdown, followed by shRNA-mediatedknockdown to investigate long-term depletion of the target genes. Panel(B) is a table listing the names and sequences of the siRNAs and shRNAsused in this study.

FIG. 2 shows VALIDATION OF TARGET GENES VIA QPCR

(A) and (B), CHO-DG44 cells stably secreting the IgG1 antibody mAb1 weretransfected with microRNA miR-c #1, miR-1287 or miR-1978. One day aftertransfection, RNA was extracted to quantify levels of mRNA of ATF6B,CerS2 and Tbc1D20 by qPCR. (C) and (D), Expression levels of thesetarget genes were quantified for CHO-DG44 cells stably secreting theantibody mAb1 and further stably overexpressing either miR-1287 ormiR-1978. Relative expression was calculated by normalizing to thereference gene beta actin. Panel (A) is a bar graph showing thattransient transfection with miR-1287 resulted in a reduced expressionlevel of ATF6B compared to untransfected cells (mock) (n=3, errorbars=SEM of triplicates). Panel (B) is a bar graph showing thattransient transfection with the miRNA miR-1978 resulted in decreasedlevels of CERS2 mRNA and TBC1D20 mRNA compared to untransfected cells(mock) (n=3, error bars=SEM of triplicates). (C)-(D) CHO-mAb1 cellsstably secreting an IgG antibody were stably transfected with expressionvectors encoding either miR-1287 or miR-1978(pcDNA6.2-GW/emGFP-miR1287-miR1287 or pcDNA6.2-GW/emGFP-miR1978-miR1978)or a negative control sequence expression plasmid(pcDNA6.2-GW/emGFP-negative-control) and enriched for GFP positive cellsby FACS. Panel (C) is a bar graph showing that CHO-mAb1 cells stablyoverexpressing miR-1287 showed reduced levels of ATF6B mRNA compared toparental CHO-mAb1 cells (n=3, error bars=SEM of triplicates). Panel (D)is a bar graph showing that CHO-mAb1 cells stably overexpressingmiR-1978 showed reduced levels of both CERS2 and TBC1D20 compared toparental CHO-mAb1 cells (n=2, error bars=SEM of duplicates).

FIG. 3 shows KNOCKDOWN EFFICIENCY OF SIRNAS SPECIFIC FOR ATF6B, CERS2AND TBC1D20

CHO-DG44 cells stably secreting the IgG1 antibody mAb1 were transfectedwith siRNAs specific for ATF6B or CERS2 and TBC1D20, respectively, andmRNA levels of ATF6B, CERS2 and TBC1D20 were quantified by qPCR. Panel(A) is a bar graph showing that an effective knockdown of ATF6B wasobserved by three independent siRNAs (siAft6b #1, siAft6b #2 and siAft6b#3) (n=3, error bars=SEM of triplicates). Panel (B) is a bar graphshowing that combined knockdown of CERS2 and TBC1D20 was observed bynucleofection with both siRNAs simultaneously (siTbc1D20 #1 and siCerS2#1) (n=2, error bars=SEM of duplicates).

FIG. 4 shows the EFFECT OF ATF6B KNOCKDOWN ON PRODUCTIVITY OF CHO-mAb1CELLS

CHO-mAb1 cells were transfected with each of three independent siRNAsspecific for ATF6B (siAft6b #1, siAft6b #2 and siAft6b #3). Specificproductivity on days 3 and 4 was calculated and normalized tonon-transfected control cells (mock). Knockdown of ATF6B resulted inimproved specific productivity at day 4 (n=3, error bars=SEM oftriplicates).

FIG. 5 shows the EFFECT OF COMBINED CERS2 AND TBC1D20 KNOCKDOWN ONPRODUCTIVITY OF CHO-mAB1 CELLS

CHO-mAb1 cells were transfected with siRNAs against CERS2 and TBC1D20(siTbc1D20 #1 and siCerS2 #1) (A) singly and (B) in combination.Specific productivity on days 3 and 4 was calculated and normalized tonon-transfected control cells (mock). Panel (A) is a bar graph showingthat a single knockdown of either CERS2 or TBC1D20 slightly increasedthe specific productivity at day 4 (n=2, error bars=SEM of duplicates).Panel (B) is a bar graph showing that a combined knockdown of CERS2 andTBC1D20 resulted in further improvement of the specific productivity atday 3 and day 4 (n=3, error bars=SEM of triplicates).

FIG. 6 shows the EFFECT OF ATF6B KNOCKDOWN ON PRODUCTIVITY OF CHO-mAB2CELLS

A further CHO-DG44 cell clone stably secreting the IgG antibody mAb2 wastransfected with each of three independent siRNAs specific for ATF6B(siAft6b #1, siAft6b #2 and siAft6b #3). Specific productivity on days 3and 4 was calculated and normalized to non-transfected control cells(mock). Knockdown of ATF6B resulted in improved specific productivity atdays 3 and 4 (n=3, error bars=SEM of replicates).

FIG. 7 shows the EFFECT OF COMBINED CERS2 AND TBC1D20 KNOCKDOWN ONPRODUCTIVITY OF CHO-mAB2 CELLS

CHO-DG44 cells stably secreting the IgG antibody mAb2 were transfectedwith siRNAs against CERS2 and TBC1D20 in combination (siTbc1D20 #1 andsiCerS2 #1). Specific productivity on days 3 and 4 was calculated andnormalized to non-transfected control cells (mock). Combined knockdownof CERS2 and TBC1D20 resulted in improved specific productivity at day 3and day 4 (n=3, error bars=SEM of triplicates).

FIG. 8 shows QUANTIFICATION OF STABLE GFP EXPRESSION IN ANTIBODYPRODUCING CHO CELLS BY FLOW CYTOMETRY

CHO-DG44 cells stably secreting the IgG antibody mAb2 were stablytransfected with a GFP-containing expression vector further encoding (asshown in panel A) a shRNA specific for ATF6B (pcDNA6.2-GW/emGFP-shATF6B#1 whose sequence structure is schematically represented in panel B),and (as show in panel C) a shRNA specific for CERS2 and for TBC1D20(pcDNA6.2-GW/emGFP-shTBC1D20 #1-shCERS2 #1 or pcDNA6.2-GW/emGFP-shCERS2#2-shTBC1D20 #1, whose sequence structures are schematically representedpanel D). GFP-positive cells were enriched by FACS and living cells wereanalyzed by flow cytometry analysis after cultivation for 42 days aftersorting (see panels A and C). As a negative control, untransfected cellswithout GFP expression were used (greyed out).

FIG. 9 shows QUANTIFICATION OF STABLE KNOCKDOWN IN ANTIBODY PRODUCINGCHO CELLS BY QPCR

CHO-DG44 cells stably secreting the IgG antibody mAb2 were stablytransfected with a GFP-containing expression vector further encoding (asshown in panel A) a shRNA specific for ATF6B (shAtf6b #1) or (as shownin panel B) two combined shRNAs specific for CERS2 and TBC1D20 (shCerS2#1-shTbc1D20 #1, shCerS2 #2-shTbc1D20 #1). GFP-positive cells wereenriched by FACS sorting and RNA was extracted to measure levels of mRNAof ATF6B or CERS2 and TBC1D20, respectively, by qPCR analysis. Relativeexpression was calculated by normalizing to the reference gene betaactin.

FIG. 10 shows the EFFECTS OF STABLE KNOCKDOWN OF ATF6B IN FED-BATCH CELLCULTURE

CHO-DG44 cells stably secreting the IgG antibody mAb2 were stablytransfected with shRNAs against ATF6B (pcDNA6.2-GW/emGFP-shAtf6b #1) ora negative control sequence expression plasmid(pcDNA6.2-GW/emGFP-negative-control) and enriched for GFP positive cellsby FACS sorting. Stable pools (one pool for shAtf6b #1 and twoindependent pools for the negative control plasmid) were grown infed-batch cultures. Cell density and antibody concentration in thesupernatant were determined on days 3-6 by cell counting with trypanblue exclusion and ELISA analysis, respectively, and specificproductivity was calculated. The data for one representative experimentare shown for the product concentration (in panel A), the specificproductivity at day 6 (in panel B), and the viable cell density (inpanel C).

FIG. 11 shows the EFFECTS OF STABLE KNOCKDOWN OF CERS2 AND TBC1D20 INFED-BATCH CELL CULTURE

CHO-DG44 cells stably secreting the IgG antibody mAb2 were stablytransfected with expression vectors encoding for shRNAs specific forCERS2 and TBC1D20 (pcDNA6.2-GW/emGFP-shTbc1D20 #1-shCerS2 #1 andpcDNA6.2-GW/emGFP-shCerS2 #2-shTbc1D20 #1) or a negative controlsequence (pcDNA6.2-GW/emGFP-negative-control) and enriched for GFPpositive cells by FACS sorting. Stable pools (one pool for each of twoindependent shRNA combinations and 2 independent pools for the negativecontrol plasmid) were grown in fed-batch cultures. Cell density andantibody concentration in the supernatant were determined on days 3-11by cell counting with trypan blue exclusion and ELISA analysis,respectively, and specific productivity was calculated. The data for onerepresentative experiment are shown for the product concentration (inpanel A), the specific productivity at day 11 (in panel B) and theviable cell density (in panel C).

FIG. 12 shows the ANALYSIS OF ANTIBODY GLYCOSYLATION AND AGGREGATEFORMATION UNDER FED-BATCH CONDITIONS

CHO-DG44 cells stably secreting the IgG antibody mAb2 were stablytransfected with expression vectors expressing (in panels A, C) a shRNAspecific for ATF6B (pcDNA6.2-GW/emGFP-shAtf6b #1) or (in panels B,D)shRNAs specific for CERS2 and TBC1D20 (pcDNA6.2-GW/emGFP-shTbc1D20#1-shCerS2 #1 and pcDNA6.2-GW/emGFP-shCerS2 #2-shTbc1D20 #1). As acontrol the same antibody producing cells were transfected with anegative control sequence expression plasmid(pcDNA6.2-GW/emGFP-negative-control). (A, B) The composition of theFc-glycosylation of the IgG antibody was analyzed after PNGaseF releaseand fluorescent labelling using microchip-based capillaryelectrophoresis (CE). The percentage of the glycol-forms was calculatedfrom the chromatographic peak areas and is shown for (in panel A)shATF6b expressing and (in panel B) shCERS2 and shTBC1D20 expressingcells. The glycans in the legend on the right hand side are listedinversely to the order in each bar, i.e., Man 5 at the bottom, UNKsecond from the bottom etc. Abbreviations: A2, biantennary; G,galactose; F, fucose; Man, mannose; UNK, unknown; asterisks indicateisoforms. (C) CHO-DG44 cells stably secreting the IgG antibody mAb2 andstably expressing shAtf6b #1, shAtf6b #2 or a negative control sequencewere cultivated under fed-batch conditions for 7 days. The secretedantibody was purified from the supernatants and analyzed by HPLC and theabsorption over time of the three samples analysed is shown in panel C.(D) CHO-mAb2 cells stably expressing shTbc1D20-shCerS2 #1, shCerS#2-shTbc1D20 or a negative control sequence were cultivated underFed-batch conditions for 11 days. The secreted antibody was purifiedfrom the supernatant and analyzed by HPLC and the absorption over timeof the three samples analysed is shown in panel D.

FIG. 13 shows the EFFECT OF ATF6B KNOCKDOWN ON THE EXPRESSION OF GRP78,CHOP AND HERPUD1 AFTER TUNICAMYCIN TREATMENT

CHO-DG44 cells stably secreting the IgG antibody mAb2 were stablytransfected with a plasmid encoding a GFP cassette plus a shRNA sequencecomprising a nucleotide sequence specifically targeting ATF6B(pcDNA6.2-GW/emGFP-shAtf6b #1 or pcDNA6.2-GW/emGFP-shAtf6b #2), or anegative control construct. Panel A is a bar graph showing the mRNAlevel of Atf6b as quantified by qPCR. Values are given relative tocontrol. Panel B provides bar graphs showing the mRNA levels of GRP78,Herpud1 and CHOP in untreated cells and tunicamycin™ treated cells (2.5μg/mL TM for 6 hours) as quantified by qPCR and shown relative tonegative control cells at time point 0. All data are shown as mean+/−SEM(n=3, **p<0.01, ***p<0.001, One Way Anova and Tukeys Multiple ComparisonTest), representing negative control (black bar, left); shAtf6b #1 (greybar, middle); and shAtf6b #2 (white bar, right) as also shown in thelegend of FIG. 13C. (C) CHO-DG44 cells stably secreting the IgG antibodymAb2 and stably expressing shAtf6b #1, shAtf6b #2 or a negative controlsequence were cultivated under fed-batch conditions for 5 days. At day 5the mRNA levels of GRP78/BiP, Herpud1 and CHOP were quantified by qPCR(as shown in panel C). All data are shown as mean+/−SEM (N=3, **p<0.01,Two Way Anova and Bonferroni posttest).

FIG. 14 shows the EFFECT OF CERS2 AND TBC1D20 KNOCKDOWN ON RAB1 ACTIVITYAND CERAMIDE COMPOSITION

(A) CHO-DG44 cells stably secreting the IgG antibody mAb2 weretransfected with a non-targeting siRNA (NT) as a control, siTbc1D20 orsiCerS2/siTbc1D20. Three days post transfection active Rab1 was detectedin cell homogenates using a pull-down assay with a p115-GST fusionprotein (as shown in panel A). Bound Rab1 was detected by immunoblottingwith an anti-Rab1 antibody. An anti-GST antibody was used to detect thep115-GST protein as loading control. Detection of actin in the lysatesverifies equal loading of protein (input). Panel B is a bar graphshowing that the band intensity of active Rab1 was quantified usingImage J software. (n=3, mean±SEM). Panel C shows that knockdownefficiency was confirmed by qPCR at day 2 post transfection (n=3,mean±SEM). (D) Three days after transfection with NT siRNA control,siCerS2 or siTbc1D20/siCerS2, cells were homogenized and ceramidesynthase activity was measured by adding either C16-CoA or C24-CoA assubstrates together with NBD-labeled sphinganine. The productsC16-dihydroceramide and C24-dihydroceramide were separated by thin layerchromatography and detected using a fluorescence scanner (as shown inpanel D). Panel E is a bar graph showing band intensities of C16- andC24-dihydroceramides as quantified using Image J software. Theintensities of both products in cells transfected with the siRNA controlwere set to 100% (n=3, mean±SEM). Panel F is a bar graph showing thatthe efficiency of siRNA-mediated knockdown was confirmed by qPCR (n=2,mean±SEM).

FIG. 15 shows the EFFECTS OF STABLE KNOCKDOWN ON DOWNSTREAM TARGETS

(A) CHO-DG44 cells stably secreting the IgG antibody mAb2 were stablytransfected with expression vectors encoding for shRNAs specific forCERS2 and TBC1D20 (pcDNA6.2-GW/emGFP-shTbc1D20 #1-shCerS2 #1 andpcDNA6.2-GW/emGFP-shCerS2 #2-shTbc1D20 #1) or a negative controlsequence (pcDNA6.2-GW/emGFP-negative-control) and were analysed in afluorescent CerS assay to detect ceramide synthase activity in CHO-mAb2cell pools. In distinct reactions, either C16-CoA or C24-CoA served assubstrates together with NBD-labeled sphinganine. The productsC16-ceramide and C24-ceramide were separated by thin layerchromatography and detected using a fluorescence scanner (as shown inpanel A). Panel B shows the band intensities of C16- and C24-ceramidesas quantified using the Image J software. The intensities of bothproducts in the negative control pool were set to 100%.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The general embodiments “comprising” or “comprised” encompass the morespecific embodiment “consisting of”. Furthermore, singular and pluralforms are not used in a limiting way. As used herein, the singular forms“a”, “an” and “the” designate both the singular and the plural, unlessexpressly stated to designate the singular only.

The term “ribonucleic acid”, “RNA” or “RNA oligonucleotide” as usedherein describes a molecule consisting of a sequence of nucleotides,which are built of a nucleobase, a ribose sugar, and a phosphate group.RNAs are usually single stranded molecules and can exert variousfunctions. The term ribonucleic acid specifically comprises messengerRNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interferingRNA (siRNA), small hairpin RNA (shRNA) and micro RNA (miRNA), each ofwhich plays a specific role in biological cells. It includes smallnon-coding RNAs, such as microRNAs (miRNA), short interfering RNAs(siRNA), small hairpin RNA (shRNA), and Piwi-interacting RNAs (piRNA).The term “non-coding” means that the RNA molecule is not translated intoan amino acid sequence.

The term “RNA interference” (RNAi) refers to sequence-specific orgene-specific suppression of gene expression (protein synthesis),without generalized suppression of protein synthesis. RNAi may involvedegradation of messenger RNA (mRNA) by an RNA-induced silencing complex(RISC), preventing translation of the transcribed mRNA. The suppressionof gene expression caused by RNAi may be transient or it may be morestable, even permanent. RNAi may be mediated by siRNA or shRNA.Preferably the RNAi according to the invention is gene-specific (onlyone gene is targeted). RNAi is considered to be gene-specific orspecific for its target gene, if the RNA oligonucleotide comprises asequence which has complete sequence complementarity with the targetgene (i.e., perfect base pairing between the antisense strand of the RNAduplex of the small interfering RNA and the target mRNA). Gene-specificRNAi may be mediated by siRNA or shRNA. In a preferred embodiment, theRNAi is mediated by siRNA or shRNA. In another preferred embodiment, theRNAi is not mediated by miRNA.

The terms “microRNA” or “miRNA” are used interchangeably herein.microRNAs are small, about 22 nucleotide-long (typically between 19 and25 nucleotides in length) non-coding single stranded RNAs. miRNAstypically target more than one gene. microRNAs are encoded in the genomeof eukaryotic cells and are typically transcribed by RNA Polymerase IIIas long primary transcripts that are then processed in several stepsfirst into ˜70 nt-long hairpin-loop structures and subsequently into the˜22 nt RNA duplex. The active mature strand is then loaded into theRNA-induced silencing complex (RISC) in order to block translation oftarget proteins or degradation of their respective mRNAs. Targeting withmiRNAs allows for mismatches and mRNA translational repression ismediated by incomplete complementarity (i.e., imperfect base paringbetween the antisense strand of the RNA duplex of the small interferingRNA and the target mRNA), while siRNA and shRNA are specific for theirtargets due to complete sequence complementarity (i.e., perfect basepairing between the antisense strand of the RNA duplex of the smallinterfering RNA and the target mRNA). Typically, miRNAs bind in the3′untranslated region (3′UTR) and are not gene-specific, but targetmultiple mRNAs. The term “microRNA” as used herein relates to endogenousgenomic mammalian miRNAs, such as human miRNAs. The prefix “hsa”indicates, e.g., the human origin of a microRNA. They may be introducedinto a mammalian host cell using an expression vector comprising genomicmicroRNA sequence(s) for transient or stable expression of miRNA in themammalian host cell. Means for cloning genomic microRNA into anexpression vector are known in the art. They include, cloning genomicmiRNA sequences with approximately 300 bp flanking regions into amammalian expression vector, such as pBIP-1, operably linked to apromoter. Alternatively one or more microRNAs may be cloned aspolynucleotides encoding engineered pre-miRNA sequences (i.e., shorthairpins) into a mammalian expression vector. For example, a maturemiRNA sequence may be cloned into a given sequence encoding an optimizedhairpin loop sequence and 3′ and 5′ flanking regions, such as derivedfrom the murine miRNA mir-155 (Lagos-Quintana et al., 2002. Curr Biol.30; 12(9):735-9). A DNA oligonucleotide is designed, which encodes themiRNA sequence, the mentioned loop and the antisense sequence of therespective mature miRNA with a two nucleotide depletion to generate aninternal loop in the hairpin stem. Furthermore, overhangs are added forcloning at both ends to fuse the DNA oligonucleotide to the 3′ and 5′flanking regions. miRNAs as used herein further comprise non-canonicalmiRNAs. These RNAs can be derived from ‘housekeeping’ non-coding RNAs(ncRNA) including ribosomal RNA (rRNA) or transfer RNA (tRNA) andfunction in a miRNA-like manner. These RNAs can also originate frommammalian mitochondrial ncRNAs and are termed mitochondrialgenome-encoded small RNAs (mitosRNAs).

As used herein, the terms “small interfering” or “short interfering RNA”or “siRNA” refer to an RNA duplex of nucleotides that is targeted to adesired gene and is capable of inhibiting the expression of a gene withwhich it shares homology. It is formed from long double stranded RNA(dsRNA) or shRNA. The RNA duplex typically comprises two complementarysingle-stranded RNAs of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29nucleotides that form 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 basepairs and possess 3′ overhangs of two nucleotides, preferably the RNAduplex comprises two complementary single stranded RNAs of 19-27nucleotides that form 17-25 base pairs and possess 3′ overhangs of twonucleotides. siRNA is “targeted” to a gene, wherein the nucleotidesequence of the duplex portion of the siRNA is complementary to anucleotide sequence of the mRNA of the targeted gene. The siRNA or aprecursor thereof is always exogenously introduced into the cell, e.g.,directly or by transfection of a vector having a sequence encoding saidsiRNA, and the endogenous miRNA pathway is harnessed for correctprocessing of siRNA and cleavage or degradation of the target mRNA. Theduplex RNA can be expressed in a cell from a single construct.

As used herein, the term “shRNA” (small hairpin RNA) refers to an RNAduplex wherein a portion of the siRNA is part of a hairpin structure(shRNA). The shRNA can be processed intracellularly into a functionalsiRNA. In addition to the duplex portion, the hairpin structure maycontain a loop portion positioned between the two sequences that formthe duplex. The loop can vary in length. In some embodiments the loop is4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 nucleotides in length. Thehairpin structure can also contain 3′ or 5′ overhang portions. In someaspects, the overhang is a 3′ or a 5′ overhang of 0, 1, 2, 3, 4 or 5nucleotides in length. In one aspect of this invention, a nucleotidesequence comprised in the vector serves as a template for the expressionof a small hairpin RNA, comprising a sense region, a loop region and anantisense region. Following expression the sense and antisense regionsform a duplex. shRNA is always exogenously introduced, e.g., bytransfection of a vector having a sequence encoding said shRNA, and theendogenous miRNA pathway is harnessed for correct processing of thesiRNA and cleavage or degradation of the target mRNA. Use of a vectorhaving a sequence encoding a shRNA has the advantage over use ofchemically synthesized siRNA in that the suppression of the target geneis typically long-term and stable.

Typically siRNA and shRNA mediate mRNA repression by complete sequencecomplementarity (i.e., perfect base paring between the antisense strandof the RNA duplex of the small interfering RNA and the target mRNA) andare therefore specific for their target. The antisense strand of the RNAduplex may also be referred to as active strand of the RNA duplex.Complete sequence complementarity of perfect base paring as used hereinmeans that the antisense strand of the RNA duplex of the smallinterfering RNA has at least 89% sequence identity with the target mRNAfor at least 15 continuous nucleotides, at least 16 continuousnucleotides, at least 17 continuous nucleotides, at least 18 continuousnucleotides and preferably at least 19 continuous nucleotides, orpreferably at least 93% sequence identity with the target mRNA for atleast 15 continuous nucleotides, at least 16 continuous nucleotides, atleast 17 continuous nucleotides, at least 18 continuous nucleotides andpreferably at least 19 continuous nucleotides. More preferably theantisense strand of the RNA duplex of the small interfering RNA has 100%sequence identity with the target mRNA for at least 15 continuousnucleotides, at least 16 continuous nucleotides, at least 17 continuousnucleotides, at least 18 continuous nucleotides and preferably at least19 continuous nucleotides.

The term “ATF6B” as used herein refers to the protein ActivatingTranscription Factor 6 Beta. It is also known as CREBL1, CAMP ResponseElement-Binding Protein-Related Protein, CAMP Responsive Element BindingProtein-Like 1, CAMP-Dependent Transcription Factor ATF-6 Beta,CAMP-Responsive Element-Binding Protein-Like 1, Protein G13 CREB-RP,G13, Cyclic AMP-Dependent Transcription Factor ATF-6 Beta, Creb-RelatedProtein, ATF6-Beta. External Ids for Atf6b are: HGNC: 2349; Entrez Gene:1388; Ensembl: ENSG00000213676; OMIM: 600984; UniProtKB: Q99941(http://www.genecards.org/cgi in/carddisp.pl?gene=ATF6B&keywords=ATF6B).It comprises proteins having the amino acid sequence of SEQ ID NO: 6 oran amino acid sequence having at least 80% sequence identity with SEQ IDNO:6. The term “CERS2” as used herein refers to the protein CeramindeSynthase 2. It is also known as LASS2, Longevity Assurance (LAG1, S.Cerevisiae) Homolog 2, Tumor Metastasis-Suppressor Gene 1 Protein, LAG1Homolog, Ceramide Synthase 2, SP260, TMSG1, LAG1 Longevity AssuranceHomolog 2 (S. Cerevisiae), LAG1 Longevity Assurance Homolog 2, LAG1Longevity Assurance 2, L3. External Ids for CerS2 are: HGNC: 14076;Entrez Gene: 29956; Ensembl: ENSG00000143418; OMIM: 606920; UniProtKB:Q96G23 (http://www.genecards.org/cgi-bin/carddisp.pl?gene=CERS2). Itcomprises proteins having the amino acid sequence of SEQ ID NO: 5 or anamino acid sequence having at least 80% sequence identity with SEQ IDNO:5.

The term “TBC1D20” as used herein refers to TBC1 Domain Family Member20. It is also known as C20orf140, WARBM4 and Chromosome 20 Open ReadingFrame 140. External Ids for Tbc1d20 are: HGNC: 16133; Entrez Gene:128637; Ensembl: ENSG00000125875; OMIM: 611663; UniProtKB: Q96BZ9(http://www.genecards.org/cgi-bin/carddisp.pl?gene=TBC1D20). Itcomprises proteins having the amino acid sequence of SEQ ID NO: 4 or anamino acid sequence having at least 80% sequence identity with SEQ IDNO: 4.

As used herein, an RNA oligonucleotide that inhibits the expression ofTBC1D20, CERS2 or ATF6B by RNAi specifically targets the RNA encodingTBC1D20, CERS2 or ATF6B, respectively. In a preferred embodiment, theRNA oligonucleotide excludes miRNA.

The term “knockdown” or “knockdown technology” refers to a technique ofgene silencing in which the expression of a target gene or gene ofinterest is reduced as compared to the gene expression prior to theintroduction of an RNA oligonucleotide that inhibits expression of atarget gene by RNA-interference, such as by using siRNA or shRNA, whichcan lead to the inhibition of production of the target gene product.“Double knockdown” is the knockdown of two genes.

A gene may also be modified by deleting the gene using “knockout”technology. The term “knockout” refers to cells, which have beengenetically modified so that the expression of host cell proteins AFT6Bor the combination of host cell proteins TBC1D20 and CERS2 is/areinhibited and the respective host cell protein is not produced (reducedby 100%). This may be achieved using various technologies, which areknown in the art, including CRISPR-Cas9 or Zinc finger nucleasetechnology.

Alternatively, the gene may be altered to inhibit the expression of itsprotein by introduction of a mutation in the gene. The gene mutation maybe a deletion, addition or substitution in the coding region or in thepromoter or regulatory region of the gene. This gene mutation may beeither in one or both alleles of a gene.

The term “reduction”, “reduced” or “reduce”, as used herein, generallymeans a decrease by at least 10% as compared to a reference level, forexample a decrease by at least about 20%, or at least about 30%, or atleast about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 75%, or at least about 80%, or atleast about 90% or up to and including a 100% decrease, or any integerdecrease between 10-100% as compared to a control mammalian cell. Theexpression of the host cell proteins TBC1D20 and CERS2, or of the hostcell protein ATF6B, is preferably reduced by at least 30%, at least 40%,at least 50%, at least 75%, or 100%, compared to a control mammaliancell.

The term “enhancement”, “enhanced”, “enhanced”, “increase” or“increased”, as used herein, generally means an increase by at least 10%as compared to a control cell, for example an increase by at least about20%, or at least about 30%, or at least about 40%, or at least about50%, or at least about 75%, or at least about 80%, or at least about90%, or at least about 100%, or at least about 200%, or at least about300%, or any integer decrease between 10-300% as compared to a controlcell.

As used herein, a “control cell” or “control mammalian cell” is a cellwhich is the same as the cell to which it is compared to, except that itdoes not have reduced expression of the host cell proteins TBC1D20 andCERS2 or ATF6B. The control mammalian cell may be produced by a methodof the present invention, but omitting the step of reducing theexpression of host cell proteins TBC1D20 and CERS2, or of the host cellprotein ATF6B. In particular, the control mammalian cell lacks any agenetic modification that inhibits expression of TBC1D20 and CERS2, orATF6B and lacks any transfected RNA oligonucleotide that inhibits thegenes Tbc1d20 and Cers2, or Atf6b, by RNA-interference.

The term “derivative” or “homologue” as used in the present inventionmeans a polypeptide molecule or a nucleic acid molecule, which is atleast 70% identical in sequence with the original sequence or itscomplementary sequence. Preferably, the polypeptide molecule or nucleicacid molecule is at least 80% identical in sequence with the originalsequence or its complementary sequence. More preferably, the polypeptidemolecule or nucleic acid molecule is at least 90% identical in sequencewith the original sequence or its complementary sequence. Still morepreferably, the polypeptide molecule or a nucleic acid molecule is atleast 95% identical in sequence with the original sequence or itscomplementary sequence. Most preferably the polypeptide molecule or anucleic acid molecule is at least 98% identical in sequence with theoriginal sequence or its complementary sequence. A homologous proteinfurther displays the same or a similar protein activity as the originalsequence.

Sequence differences may be based on differences in homologous sequencesfrom different organisms or may be naturally occurring allelicvariations. They might also be based on targeted modification ofsequences by substitution, insertion or deletion of one or morenucleotides or amino acids, preferably 1, 2, 3, 4, 5, 7, 8, 9 or 10.Deletion, insertion or substitution mutants may be generated usingsite-specific mutagenesis and/or PCR-based mutagenesis techniques.

The term “host cells” as used herein are mammalian cells lines suitablefor the production of a secreted recombinant therapeutic protein and mayhence also be referred to as “mammalian cells”. Preferred mammaliancells according to the invention are rodent cells such as hamster cells.The mammalian cells are isolated cells or cell lines. The mammaliancells are preferably transformed and/or immortalized cell lines. Theyare adapted to serial passages in cell culture and do not includeprimary non-transformed cells or cells that are part of an organstructure. Preferred mammalian cells are BHK21, BHK TK⁻, CHO, CHO-K1(such as CHO-DUKX, CHO-DUKX B1) and CHO-DG44 cells or thederivatives/progenies of any of such cell line. Particularly preferredare CHO-DG44, CHO-K1 and BHK21, and even more preferred are CHO-DG44 andCHO-K1 cells. Most preferred are CHO-DG44 cells. Glutamine synthetase(GS)-deficient derivatives of the mammalian cell, particularly of theCHO-DG44 and CHO-K1 cell are also encompassed. The mammalian cell mayfurther comprise one or more expression cassette(s) encoding arecombinant secreted therapeutic protein. The host cells may also bemurine cells such as murine myeloma cells, such as NS0 and Sp2/0 cellsor the derivatives/progenies of any of such cell line. Non-limitingexamples of mammalian cells which can be used in the meaning of thisinvention are also summarized in Table 1. However, derivatives/progeniesof those cells, other mammalian cells, including but not limited tohuman, mice, rat, monkey, and rodent cell lines, can also be used in thepresent invention, particularly for the production of biopharmaceuticalproteins.

TABLE 1 Mammalian production cell lines Cell line Order Number NS0 ECACCNo. 85110503 Sp2/0-Ag14 ATCC CRL-1581 BHK21 ATCC CCL-10 BHK TK⁻ ECACCNo. 85011423 HaK ATCC CCL-15 2254-62.2 (BHK-21 derivative) ATCC CRL-8544CHO ECACC No. 8505302 CHO wild type ECACC 00102307 CHO-K1 ATCC CCL-61CHO-DUKX ATCC CRL-9096 (═CHO duk⁻, CHO/dhfr⁻) CHO-DUKX B11 ATCC CRL-9010CHO-DG44 Urlaub G, et al., 1983. Cell. 33: 405-412. CHO Pro-5 ATCCCRL-1781 V79 ATCC CCC-93 B14AF28-G3 ATCC CCL-14 HEK 293 ATCC CRL-1573COS-7 ATCC CRL-1651 U266 ATCC TIB-196 HuNS1 ATCC CRL-8644 CHL ECACC No.87111906 CAP¹ Wölfel J, et al., 2011. BMC Proc. 5(Suppl 8): P133.PER.C6 ® Pau et al., 2001. Vaccines. 19: 2716-2721. H4-II-E ATCCCRL-1548 ECACC No. 87031301 Reuber, 1961. J. Natl. Cancer Inst. 26:891-899. Pitot H. C., et al., 1964. Natl. Cancer Inst. Monogr. 13:229-245. H4-II-E-C3 ATCC CRL-1600 H4TG ATCC CRL-1578 H4-II-E DSM ACC3129H4-II-Es DSM ACC3130 ¹CAP (CEVEC's Amniocyte Production) cells are animmortalized cell line based on primary human amniocytes. They weregenerated by transfection of these primary cells with a vectorcontaining the functions E1 and pIX of adenovirus 5. CAP cells allow forcompetitive stable production of recombinant proteins with excellentbiologic activity and therapeutic efficacy as a result of authentichuman posttranslational modification.

Host cells are most preferred, when being established, adapted, andcompletely cultivated under serum free conditions, and optionally inmedia, which are free of any protein/peptide of animal origin.Commercially available media such as Ham's F12 (Sigma, Deisenhofen,Germany), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM;Sigma), Minimal Essential Medium (MEM; Sigma), Iscove's ModifiedDulbecco's Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, Calif.),CHO-S-Invitrogen), serum-free CHO Medium (Sigma), and protein-free CHOMedium (Sigma) are exemplary appropriate nutrient solutions. Any of themedia may be supplemented as necessary with a variety of compounds,non-limiting examples of which are hormones and/or other growth factors(such as insulin, transferrin, epidermal growth factor, insulin likegrowth factor), salts (such as sodium chloride, calcium, magnesium,phosphate), buffers (such as HEPES), nucleosides (such as adenosine,thymidine), glutamine, glucose or other equivalent energy sources,antibiotics and trace elements. Any other necessary supplements may alsobe included at appropriate concentrations that would be known to thoseskilled in the art. In the present invention the use of serum-freemedium is preferred, but media supplemented with a suitable amount ofserum can also be used for the cultivation of host cells. For the growthand selection of genetically modified cells expressing the selectablegene a suitable selection agent is added to the culture medium.

The term “protein” is used interchangeably with “amino acid residuesequences” or “polypeptide” and refers to polymers of amino acids of anylength. These terms also include proteins that are post-translationallymodified through reactions that include, but are not limited to,glycosylation, acetylation, phosphorylation or protein processing.Modifications and changes, for example fusions to other proteins, aminoacid sequence substitutions, deletions or insertions, can be made in thestructure of a polypeptide while the molecule maintains its biologicalfunctional activity. For example certain amino acid sequencesubstitutions can be made in a polypeptide or its underlying nucleicacid coding sequence and a protein can be obtained with the sameproperties.

The term “polypeptide” means a sequence with more than 10 amino acidsand the term “peptide” means sequences with up to 10 amino acids inlength. However, the terms may be used interchangeably.

The present invention is suitable to generate host cells for theproduction of biopharmaceutical or diagnostic polypeptides/proteins. Theinvention is particularly suitable for the high-yield expression of alarge number of different genes of interest by cells showing enhancedcell productivity.

“Recombinant secreted therapeutic protein” refers to a secreted proteinof interest suitable for diagnostic or therapeutic use, preferably fortherapeutic use encoded by a polynucleotide sequence of any length thathas been introduced into a host cell. The selected sequence encoding theprotein can be full length or a truncated gene, a fusion or tagged gene,and can be a cDNA, a genomic DNA, or a DNA fragment, preferably a cDNA.It can be the native sequence, i.e. naturally occurring form(s), or canbe mutated or otherwise modified as desired. These modifications includecodon optimizations to optimize codon usage in the selected host cell,humanization, fusion or tagging. A “recombinant” protein is a proteinexpressed from a heterologous sequence.

The term “host cell protein” generally relates to all proteinsendogenous to the host cell, but is used herein as specifically relatingto the host cell protein ATF6B or to the two host cell proteins TBC1D20and CERS2.

The term “producing” or “highly producing”, “production”, “productionand/or secretion”, “producing” or “production cell” as used hereinrelates to the production of the recombinant secreted therapeuticprotein. An “increased production and/or secretion” relates to theexpression of the recombinant secreted therapeutic protein and means anincrease in specific productivity, increased titer or both. Preferably,the titer or the specific productivity and the titer are increased.Increased titer as used herein relates to an increased concentration inthe same volume, i.e., an increase in total yield. The producedrecombinant secreted therapeutic protein may be, for example, anantibody, preferably a monoclonal antibody, a bispecific antibody or afragment thereof, or a fusion protein, preferably a Fc-fusion protein.

As used herein, the term “expression cassette” refers to the part of avector comprising one or more genes encoding for a protein (recombinantsecreted therapeutic protein) and the sequences controlling theirexpression. Thus it comprises a promoter sequence, an open reading frameand a 3′ untranslated region, typically containing a polyadenylationsite. Preferably the vector is an expression vector comprising one ormore gene encoding for the recombinant secreted therapeutic protein. Itmay be part of a vector, typically an expression vector, including aplasmid or a viral vector. It may also be integrated in a chromosome byrandom or targeted integration, such as by homologous recombination. Anexpression cassette is prepared using cloning techniques and doestherefore not refer to a natural occurring gene structure.

A “vector” is a nucleic acid that can be used to introduce anothernucleic acid (or “construct”) linked to it into a cell. One type ofvector is a “plasmid”, which refers to a linear or circular doublestranded DNA molecule into which additional nucleic acid segments can beligated. Another type of vector is a viral vector (e.g., replicationdefective retroviruses, adenoviruses and adeno-associated viruses),wherein additional DNA segments can be introduced into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors comprising abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) integrate into the genomeof a host cell upon introduction into the host cell and culturing underselective pressure, and thereby are replicated along with the hostgenome. A vector can be used to direct the expression of a chosenpolynucleotide in a cell.

The term “expression vector” means a nucleic acid that has the abilityconfer expression of a nucleic acid fragment to which it is operablylinked in a cell. As used herein, an expression vector may be forexample a plasmid, a cosmid, virus sub-genomic or genomic fragment, orother nucleic acid capable of maintaining and/or replicatingheterologous DNA in an expressible format in a mammalian cell.

The term “antibody” refers to a protein consisting of one or morepolypeptides substantially encoded by immunoglobulin genes. Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant regions genes as well as the myriadimmunoglobulin variable region genes. As used herein, the term“antibody” includes a polyclonal, monoclonal, bi-specific,multi-specific, human, humanized, or chimeric antibody. The terms“antibody” and “immunoglobulin” are used interchangeably and are used todenote, without being limited thereto, glycoproteins having thestructural characteristics noted above for immunoglobulins.

The term “antibody” is used herein in its broadest sense and encompassesmonoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, chimeric antibodies, humanized antibodies, humanantibodies, multispecific antibodies (e.g. bispecific antibodies),single domain antibodies, and antibody fragments (such as Fv, Fab, Fab′,F(ab)2 or other antigen-binding subsequences of antibodies). The term“antibody” also encompasses antibody conjugates and fusion antibodies.Bispecific antibodies include BITE® (Bispecific T-cell Engager) andDART® (Dual-Affinity Re-Targeting) antibodies. Single domain antibodiesinclude camelids antibodies. Full length “antibodies” or“immunoglobulins” are generally heterotetrameric glycoproteins of about150 kDa, composed of two identical light and two identical heavy chains.Each light chain is linked to a heavy chain by one covalent disulphidebond, while the number of disulphide linkages varies between the heavychains of different immunoglobulin isotypes. Each heavy and light chainalso has regularly spaced intrachain disulphide bridges. Each heavychain has an amino terminal variable domain (VH) followed by threecarboxy terminal constant domains (CH). Each light chain has a variableN-terminal domain (VL) and a single C-terminal constant domain (CL). Theterm “antibody” further refers to a type of antibody comprising aplurality of individual antibodies having the same specificity (variabledomain) and having the same constant domains.

The term “monoclonal antibody” (mAb) as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies based on the amino acid sequence. Monoclonal antibodies arehighly specific, being directed against a single antigenic site.Furthermore, in contrast to conventional (polyclonal) antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each mAb is directed againsta single determinant on the antigen. In addition to their specificity,the mAbs are advantageous in that they can be synthesized by cellculture (hybridomas, recombinant cells or the like) uncontaminated byother immunoglobulins. The mAbs herein include chimeric, humanized andhuman antibodies.

“Chimeric antibodies” are antibodies, wherein light and/or heavy chaingenes have been constructed, typically by genetic engineering, fromimmunoglobulin variable and constant regions of different species, suchas mouse and human. Or alternatively, whose heavy chain genes arebelonging to a particular antibody class or subclass while the remainderof the chain is from another antibody class or subclass of the same oranother species. Also covered are fragments of such antibodies,preferably fragments that contain or are modified to contain at leastone CH2 domain. For example, the variable segments of the genes from amouse monoclonal antibody may be joined to human constant segments, suchas gamma 1 and gamma 3. A typical therapeutic chimeric antibody is thusa hybrid protein composed of the variable or antigen-binding domain froma mouse antibody and the constant or effector domain from a humanantibody (e.g. ATCC Accession No. CRL 9688 secretes an anti-Tac chimericantibody), although other mammalian species may be used.

The term “humanized antibodies” as used herein refers to specificchimeric antibodies, immunoglobulin chains or fragments thereof (such asFv, Fab, Fab′, F(ab)2 or other antigen-binding subsequences ofantibodies), which contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies comprise a human framework regionand one or more CDRs from a non-human (usually a mouse or rat) antibody.Preferably they contain or are modified to contain at least the portionof the CH2 domain of the heavy chain immunoglobulin constant regioncomprising the N-linked glycosylation site. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a complementary-determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. Adjustments in framework amino acids might berequired to keep antigen binding specificity, affinity and or structureof domain. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by the corresponding non-human residues.Furthermore, humanized antibodies can comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and maximizeantibody performance. In general, the humanized antibody will compriseat least one, and typically two, variable domains, in which all orsubstantially all off the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the framework regions arethose of a human immunoglobulin consensus sequence. Preferably, thehumanized antibody also comprises at least a portion of animmunoglobulin constant region, typically that of a humanimmunoglobulin.

The term “CH2 domain” according to the present invention is meant todescribe the CH2 domain of the heavy chain immunoglobulin constantregion comprising the N-linked glycosylation site. In defining animmunoglobulin CH2 domain reference is made to immunoglobulins ingeneral and in particular to the domain structure of immunoglobulins asapplied to human IgG1 by Kabat, E. A. (Kabat E A, 1988. J. Immunol.141:S25-S36; Kabat E A, et al., 1991. Sequences of Proteins ofImmunological Interest. U. S. Department of Health and Human Services,Natl. Inst. of Health, Bethesda). Accordingly, immunoglobulins aregenerally heterotetrameric glycoproteins of about 150 kDa, composed oftwo identical light and two identical heavy chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulins isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has an aminoterminal variable domain (VH) followed by carboxy terminal constantdomains (CH). Each light chain has a variable N-terminal domain (VL) anda C-terminal constant domain (CL).

Depending on the amino acid sequence of the constant domain of the heavychains, antibodies can be assigned to different classes. There are fivemajor classes: IgA, IgD, IgE, IgG and IgM. The heavy chain constantdomains that correspond to the different classes of antibodies arecalled alpha, delta, epsilon, gamma and mu domains, respectively. The muchain of IgM contains five domains (VH, CHmu1, CHmu2, CHmu3 and CHmu4).The heavy chain of IgE also contains five domains while the heavy chainof IgA has four domains. The immunoglobulin class can be further dividedinto subclasses (isotypes), e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

The subunit structures and three-dimensional configuration of differentclasses of immunoglobulins are well known. Of these IgA and IgM arepolymeric and each subunit contains two light and two heavy chains. Theheavy chain of IgG contains a polypeptide chain lying between theCHgamma1 and CHgamma2 domains known as the hinge region. The alpha chainof IgA has a hinge region containing an O-linked glycosylation site. Themu and epsilon chains do not have a sequence analogous to the hingeregion of the gamma and alpha chains, however, they contain a fourthconstant domain lacking in the other in the other immunoglobulinclasses.

The Fc region of a full antibody usually comprises two CH2 domains andtwo CH3 domains. According to the present invention, the CH2 domain ispreferably the CH2 domain of one of the five immunoglobulin classesindicated above. Preferred are mammalian immunoglobulin CH2 domains suchas primate or murine immunoglobulin with the primate and especiallyhuman immunoglobulin CH2 domains being preferred. The amino acidsequences of immunoglobulin CH2 domains are known or are generallyavailable to the skilled artisan (Kabat E A, et al., 1991. Sequences ofProteins of Immunological Interest. U. S. Department of Health and HumanServices, Natl. Inst. of Health, Bethesda). A preferred immunoglobulinCH2 domain within the context of the present invention is a human IgGand preferably from IgG1, IgG2, IgG3, IgG4, more preferably a human IgG1and IgG3 and even more preferred a human IgG1. Using the numberingsystem of Edelman (Edelman G M, et al., 1969. Proc. Natl. Acad. Sci.63:78-85), the immunoglobulin CH2 domain preferably begins at amino acidposition equivalent to glutamine 233 of human IgG1 and extends throughamino acid equivalent to lysine 340 (Ellison J and Hood L, 1982. Proc.Natl. Acad. Sci. 79:1984-1988).

With respect to human antibody molecules reference is made to the IgGclass in which an N-linked oligosaccharide is attached to the amide sidechain of Asn 297 of the beta-4 bend to the inner face of the CH2 domainof the Fc region. Preferably, the antibody or Fc-fusion protein containsor is modified to contain at least a CH2 domain. The CH2 domain is a CH2domain of an immunoglobulin having a single N-linked oligosaccharide ofa human IgG CH2 domain. The CH2 domain is preferably the CH2 domain ofhuman IgG1.

“Fc-fusion proteins” are defined as proteins which contain or aremodified to contain at least the portion of the CH2 domain of the heavychain immunoglobulin constant region comprising the single N-linkedglycosylation site. According to the Kabat EU nomenclature (Kabat E A,et al., 1991. Sequences of Proteins of Immunological Interest. U. S.Department of Health and Human Services, Natl. Inst. of Health,Bethesda) this N-linked glycosylation site is at position Asn297 in anIgG1, IgG2, IgG3 or IgG4 antibody. The other part of the fusion proteincan be the complete sequence or any part of the sequence of a natural ormodified heterologous protein or a composition of complete sequences orany part of the sequence of a natural or modified heterologous protein.Fc-fusion proteins can be constructed by genetic engineering approachesby introducing the CH2 domain of the heavy chain immunoglobulin constantregion comprising the N-linked glycosylation site into anotherexpression construct comprising for example other immunoglobulindomains, enzymatically active protein portions, or effector domains.Thus, an Fc-fusion protein according to the present invention comprisesalso a single chain Fv fragment linked to the CH2 domain of the heavychain immunoglobulin constant region comprising e.g. the N-linkedglycosylation site.

Furthermore, antibody fragments include e.g. “Fab fragments” (Fragmentantigen-binding=Fab). Fab fragments consist of the variable regions ofboth chains, which are held together by the adjacent constant region.These may be formed by protease digestion, e.g. with papain, fromconventional antibodies, but similar Fab fragments may also be producedby genetic engineering. Further antibody fragments include F(ab′)2fragments, which may be prepared by proteolytic cleavage with pepsin.

By definition any sequences or genes introduced into a host cell arecalled “heterologous sequences”, “heterologous genes”, “heterologousRNAs” or “transgenes” or “recombinant gene” with respect to the hostcell, even if the introduced sequence, RNA or gene is identical to anendogenous sequence, RNA or gene in the host cell. A “heterologous” or“recombinant” protein or RNA is thus a protein or RNA expressed from aheterologous sequence or gene. In a preferred embodiment, the introducedsequence, RNA or gene is not identical to an endogenous sequence, RNA orgene of the host cell in question, although embodiments where it isidentical are also contemplated in connection with the presentinvention.

“Heterologous gene” or “heterologous sequences” can be introduced into atarget cell directly (e.g., siRNAs) or by using an “expression vector”,preferably a mammalian expression vector. Methods used to constructvectors are well known to the person skilled in the art and described invarious publications. In particular techniques for constructing suitablevectors, including a description of the functional components such aspromoters, enhancers, termination and polyadenylation signals, selectionmarkers, origins of replication, and splicing signals, are reviewed inconsiderable details in (Sambrook J, et al., 1989. Molecular Cloning: ALaboratory Manual. Cold Spring Harbor: Cold Spring Harbor LaboratoryPress) and references cited therein. Vectors may include but are notlimited to plasmid vectors, phagemids, cosmids,artificial/mini-chromosomes (e.g. ACE), or viral vectors such asbaculovirus, retrovirus, adenovirus, adeno-associated virus, herpessimplex virus, retroviruses and bacteriophages. The eukaryoticexpression vectors will typically contain also prokaryotic sequencesthat facilitate the propagation of the vector in bacteria such as anorigin of replication and antibiotic resistance genes for selection inbacteria. A variety of eukaryotic expression vectors, containing acloning site into which a polynucleotide can be operably linked, arewell known in the art and some are commercially available from companiessuch as Stratagene, La Jolla, Calif.; Invitrogen, Carlsbad, Calif.;Promega, Madison, Wis. or BD Biosciences Clonetech, Palo Alto, Calif.Usually expression vectors also comprise an expression cassette encodinga selectable marker, allowing selection of host cells carrying saidexpression marker.

In the present invention the expression vectors are also used forintroducing “heterologous sequences” or “polynucleotide sequences”encoding siRNAs or shRNAs, into a host cell. Such expression vectors maycomprise siRNA or shRNA sequence(s) for transient or stable expressionof siRNA or shRNAs in cells, specifically in mammalian cells, even morespecifically in CHO cells. Preferably, said expression vector is amammalian expression vector. Means for cloning nucleotide sequencesencoding siRNAs or shRNAs into an expression vector are known to theperson skilled in the art. They include, but are not limited to cloningsiRNAs or shRNA sequences comprising flanking regions into a mammalianexpression vector, such as pcDNA6.2, or any other vector known in theart, operably linked to a promoter, preferably a strong promoter, suchas a CMV promoter or any other strong promoter known to work in the hostcell.

The term “expression” as used herein refers to transcription and/ortranslation of a heterologous nucleic acid sequence within a host cell.The level of expression of a host cell protein such as TBC1D20, CERS2 orATF6B in a host cell may be determined on the basis of either the amountof corresponding mRNA that is present in the cell, or the amount of thepolypeptide encoded by the selected sequence as in the present examples.For example, mRNA transcribed from a selected sequence can be quantifiedby Northern blot hybridization, ribonuclease RNA protection, in situhybridization to cellular RNA or by PCR, such as qPCR. Proteins encodedby a selected sequence can be quantitated by various methods, e.g. byELISA, by Western blotting, by radioimmunoassays, byimmunoprecipitation, by assaying for the biological activity of theprotein, by immunostaining of the protein followed by FACS analysis orby homogeneous time-resolved fluorescence (HTRF) assays. The level ofexpression of a non-coding RNA, such as a miRNA can also be quantifiedby PCR, such as qPCR.

The term “transformation” or “to transform”, “transfection” or “totransfect” as used herein means any introduction of genetic material,into a mammalian host cell, wherein the mammalian host cell may betransiently transfected or stably transfected. The genetic material maybe an expression vector comprising a gene of interest (e.g., arecombinant secreted therapeutic protein) or a polynucleotide sequenceencoding siRNA or shRNA. It also means the introduction of a viralnucleic acid sequence in a way which is for the respective virus thenaturally one. The viral nucleic acid sequence needs not to be presentas a naked nucleic acid sequence but may be packaged in a viral proteinenvelope.

Transfection of eukaryotic host cells with a polynucleotide orexpression vector, resulting in genetically modified cells or transgeniccells, can be performed by any method known in the art (see e.g.Sambrook J, et al., 1989. Molecular Cloning: A Laboratory Manual. ColdSpring Harbor: Cold Spring Harbor Laboratory Press). Transfectionmethods include, but are not limited to liposome-mediated transfection,calcium phosphate co-precipitation, electroporation, nucleofection,nucleoporation, microporation, polycation (such asDEAE-dextran)-mediated transfection, protoplast fusion, viral infectionsand microinjection. The transformation may result in a transient orstable transformation of the host cells. Preferably, the transfection isa stable transfection. The transfection method that provides optimaltransfection frequency and expression of the heterologous genes in theparticular host cell line and type is favoured. Suitable methods can bedetermined by routine procedures. For stable transfectants theconstructs are either integrated into the host cell's genome or anartificial chromosome/mini-chromosome or located episomally so as to bestably maintained within the host cell. Thus, the stably transfectedsequences actually remain in the genome of the cell and its daughtercells. Typically, this involves the use of a selectable marker gene andthe gene of interest or the polynucleotide sequence encoding the RNA isintegrated together with the selectable marker gene. In some cases theentire expression vector integrates into the cell's genome, in othercases only parts of the expression vector integrate into the cell'sgenome. Cells “stably expressing” a recombinant secreted therapeuticprotein or an RNA is stably transfected with a gene encoding saidrecombinant secreted therapeutic protein or with a polynucleotidesequence encoding said RNA. Thus, the sequences encoding the recombinantsecreted therapeutic protein or RNA remain in the genome of the cell andits daughter cells.

The expression vectors of the present invention may further comprise aselectable marker gene, such as an antibiotic resistance gene or anamplifiable marker gene. The amplifiable selection marker gene may beoperably linked to the polynucleotide sequence encoding the RNA. To beoperably linked, the polynucleotide sequence encoding the RNA and theamplifiable selection marker gene may be located on the same vector.Typically, the recombinant secreted therapeutic protein and thepolynucleotide sequence encoding the RNA in the expression vector of theinvention are operably linked to a promoter and/or a terminator. Therecombinant secreted therapeutic protein or the polynucleotide sequenceencoding the RNA operably linked to a promoter and/or a terminator mayalso be referred to as an expression cassette.

A “selectable marker gene” or “selection marker gene” is a gene whichencodes a selectable marker and allows the specific selection of cellswhich contain this gene, typically by the addition of a corresponding“selecting agent” to the cultivation medium. As an illustration, anantibiotic resistance gene may be used as a positive selectable marker.Only cells which have been transformed with this gene are able to growin the presence of the corresponding antibiotic and are thus selected.Untransformed cells, on the other hand, are unable to grow or surviveunder these selection conditions. There are positive, negative andbifunctional selectable markers. Positive selectable markers permit theselection and hence enrichment of transformed cells by conferringresistance to the selecting agent or by compensating for a metabolic orcatabolic defect in the host cell. By contrast, cells which havereceived the gene for the selectable marker can be selectivelyeliminated by negative selectable markers. An example of this is thethymidine kinase gene of the Herpes Simplex virus, the expression ofwhich in cells with the simultaneous addition of acyclovir organcyclovir leads to the elimination thereof. The selectable markergenes useful in this invention also include the amplifiable selectablemarkers. The literature describes a large number of selectable markergenes including bifunctional (positive/negative) markers (see forexample WO 92/08796 and WO 94/28143). Examples of selectable markerswhich are useful in the present invention include, but are not limitedto the genes of aminoglycoside phosphotransferase (APH), hygromycinephosphostransferase (HYG), dihydrofolate reductase (DHFR), thymidinekinase (TK), glutamine synthetase, asparagine synthetase and genes whichconfer resistance to neomycin (G418/Geneticin), puromycin, histidinol D,bleomycin, phleomycin, blasticidin and zeocin. Also included aregenetically modified mutants and variants, fragments, functionalequivalents, derivatives, homologues and fusions with other proteins orpeptides, provided that the selectable marker retains its selectivequalities. Such derivatives display considerable homology in the aminoacid sequence in the regions or domains, which are deemed to beselective.

Selection may also be made by fluorescence activated cell sorting (FACS)using for example a cell surface marker, bacterial β-galactosidase orfluorescent proteins (e.g. green fluorescent proteins (GFP) and theirvariants from Aequorea victoria and Renilla reniformis or other species;red fluorescent proteins, fluorescent proteins and their variants fromnon-bioluminescent species (e.g. Discosoma sp., Anemonia sp., Clavulariasp., Zoanthus sp.) to select for recombinant cells.

The term “selection agent” or “selective agent” refers to a substancethat interferes with the growth or survival of a cell, unless a certainselectable marker gene product is present in the cell which alleviatesthe effect of the selection agent. For example, to select for thepresence of an antibiotic resistance gene like APH (aminoglycosidephosphotransferase) in a transfected cell the antibiotic Geneticin(G418) is used.

The term “modified neomycin-phosphotransferase (NPT)” covers all themutants described in WO2004/050884, particularly the mutant D227G(Asp227Gly), which is characterized by the substitution of aspartic acid(Asp, D) for glycine (Gly, G) at amino acid position 227 andparticularly preferably the mutant F2401 (Phe240Ile), which ischaracterized by the substitution of phenylalanine (Phe, F) forisoleucine (Ile, I) at amino acid position 240.

The “amplifiable selectable marker gene” usually codes for an enzyme,which is needed for the growth of eukaryotic cells under certaincultivation conditions. For example, the amplifiable selectable markergene may code for dihydrofolate reductase (DHFR) or glutamine synthetase(GS). In this case the gene is amplified, if a host cell transfectedtherewith is cultivated in the presence of the selecting agentmethotrexate (MTX) or methionine sulphoximine (MSX), respectively.Sequences linked to the amplifiable selectable marker gene (i.e.,sequences physically proximal thereto) are co-amplified together withthe amplifiable selectable marker gene. Said co-amplified sequences maybe introduced on the same expression vector or on separate vectors.

The following Table 2 gives non-limiting examples of amplifiableselectable marker genes and the associated selecting agents, which maybe used according to the invention. Suitable amplifiable selectablemarker genes are also described in an overview by Kaufman (Kaufman R J,1990. Methods Enzymol. 185:537-566).

TABLE 2 Amplifiable selectable marker genes Amplifiable selectablemarker gene Accession number Selecting agent dihydrofol ate reductase(DHFR) M19869 (hamster) methotrexate (MTX) E00236 (mouse)metallothionein D10551 (hamster) cadmium M13003 (human) M11794 (rat) CAD(carbamoylphosphate M23652 (hamster) N-phosphoacetyl-L-aspartatesynthetase:aspartate D78586 (human) transcarbamylase:dihydroorotase)adenosine-deaminase K02567 (human) Xyl-A- or adenosine, M10319 (mouse)2′deoxycoformycin AMP (adenylate)-deaminase D12775 (human) adenine,azaserin, coformycin J02811 (rat) UMP-synthase J03626 (human)6-azauridine, pyrazofuran IMP 5′-dehydrogenase J04209 (hamster)mycophenolic acid J04208 (human) M33934 (mouse) xanthine-guanine- X00221(E. coli) mycophenolic acid with limiting phosphoribosyltransferasexanthine mutant HGPRTase or mutant J00060 (hamster) hypoxanthine,aminopterine and thymidine-kinase M13542, K02581 (human) thymidine (HAT)J00423, M68489(mouse) M63983 (rat) M36160 (Herpes virus)thymidylate-synthetase D00596 (human) 5-fluorodeoxyuridine M13019(mouse) L12138 (rat) P-glycoprotein 170 (MDR1) AF016535 (human) severaldrugs, e.g. adriamycin, J03398 (mouse) vincristin, colchicineribonucleotide reductase M124223, K02927 (mouse) aphidicolineglutamine-synthetase (GS) AF150961 (hamster) methionine sulphoximine(MSX) U09114, M60803 (mouse) M29579 (rat) asparagine-synthetase M27838(hamster) β-aspartylhydroxamate, albizziin, M27396 (human) 5′azacytidineU38940 (mouse) U07202 (rat) argininosuccinate-synthetase X01630 (human)canavanin M31690 (mouse) M26198 (bovine) ornithine-decarboxylase M34158(human) α-difluoromethylornithine J03733 (mouse) M16982 (rat)HMG-CoA-reductase L00183, M12705 (hamster) compactin M11058 (human)N-acetylglucosaminyl- M55621 (human) tunicamycin transferasethreonyl-tRNA-synthetase M63180 (human) borrelidin Na⁺K⁺-ATPase J05096(human) ouabain M14511 (rat)

According to the invention a preferred amplifiable selectable markergene is a gene which codes for a polypeptide with the function of GS orDHFR.

The present invention relates to mammalian cells wherein at least onegene encoding a host cell protein comprises a genetic modification thatinhibits expression of said host cell protein or the mammalian cellcomprises a RNA oligonucleotide that inhibits expression of the geneencoding a host cell protein by RNA-interference, wherein the at leastone host cell protein is ATF6B or TBC1D20 and CERS2. The invention alsorelates to methods of preparing said mammalian cells and to the use ofsaid cells in a method for producing a secreted recombinant therapeuticprotein. According to the invention, reduced expression of the host cellprotein means that the protein expression of TBC1D20 and CERS2 or theprotein expression of ATF6B in the mammalian cell is reduced compared tothe same mammalian cell not containing said genetic modification(s) orRNA oligonucleotide(s).

In one aspect, the invention relates to a mammalian cell having enhancedsecretion of a recombinant therapeutic protein comprising reducedexpression of the host cell proteins TBC1 domain family member 20(TBC1D20) and ceramide synthase 2 (CERS2); wherein the mammalian celloptionally further comprises one or more expression cassette(s) encodinga recombinant secreted therapeutic protein.

In another aspect the invention relates to a mammalian cell havingenhanced secretion of a recombinant therapeutic protein comprisingreduced expression of the host cell protein activating transcriptionfactor 6 beta (ATF6B), wherein the mammalian cell further comprises oneor more expression cassette(s) encoding a recombinant secretedtherapeutic protein.

In one embodiment of the invention, the mammalian cell having enhancedsecretion of a recombinant therapeutic protein comprises reducedexpression of the host cell proteins TBC1 domain family member 20(TBC1D20) and ceramide synthase 2 (CERS2); or reduced expression of thehost cell protein activating transcription factor 6 beta (ATF6B),wherein the mammalian cell further comprises one or more expressioncassette(s) encoding a recombinant secreted therapeutic protein.

In order to reduce expression of the host cell protein in the mammaliancell of the invention, the gene encoding the host cell protein maycomprise a genetic modification that inhibits expression of said hostcell protein, or the mammalian cell may comprise a RNA oligonucleotidethat inhibits expression of the gene encoding said host cell protein byRNA-interference. Reduced expression of the host cell protein means thatthe protein expression of TBC1D20 and CERS2 or the protein expression ofATF6B in the mammalian cell is reduced compared to the same mammaliancell not containing said genetic modification(s) or RNAoligonucleotide(s). In one embodiment, the RNA oligonucleotide thatinhibits expression of the gene of the host cell protein byRNA-interference is not a miRNA.

The invention also relates to a method of producing a mammalian cellwith enhanced secretion of a recombinant therapeutic protein comprising(a) reducing expression of the host cell proteins TBC1D20 and CERS2, orof the host cell protein ATF6B in the mammalian cell by introducing (i)a genetic modification into a gene encoding the host cell protein thatinhibits expression of said host cell protein, or (ii) a RNAoligonucleotide into the mammalian cell that inhibits expression of thegene encoding said host cell protein by RNA-interference, and (b)introducing one or more gene(s) encoding a recombinant secretedtherapeutic protein. The method may further comprise a step of (c)selecting cells with enhanced secretion of the recombinant therapeuticprotein. The method may furthermore comprise a step of (d) culturing thecells obtained in step (c) under conditions which allow expression ofone or more gene(s) encoding a recombinant secreted therapeutic protein.The introduction of one or more gene(s) encoding the secretedtherapeutic protein in step (b) preferably comprises introducing one ormore expression cassette(s) encoding the recombinant secretedtherapeutic. Step (a) of the method of the invention may be performedbefore or after step (b). Thus, the one or more gene(s) encoding therecombinant secreted protein may be introduced before the geneticmodification or RNA oligonucleotides (or the expression vectorcomprising a nucleotide sequence encoding said RNA oligonucleotide)resulting in reduced expression of the host cell protein ATF6B or thehost cell proteins TBC1D20 and CERS2 is introduced. Alternatively, theone or more gene(s) encoding the recombinant secreted protein may beintroduced after the genetic modification or RNA oligonucleotides (orthe expression vector comprising a nucleotide sequence encoding said RNAoligonucleotide) resulting in reduced the expression of the host cellprotein ATF6B or the host cell proteins TBC1D20 and CERS2 is introduced.The invention further relates to a mammalian cell line produced by themethod of the invention.

The mammalian cells and the mammalian cells produced by the method ofthe invention may further be used in a method for the production of arecombinant secreted therapeutic protein in a mammalian cell. The methodcomprising (a) providing the mammalian cell of the invention, whereinthe cell is transfected with a recombinant secreted therapeutic proteinor providing the mammalian cell produced by the method of the invention;(b) culturing the mammalian cell of step (a) in a cell culture medium atconditions allowing production of the recombinant secreted therapeuticprotein, and (c) harvesting the recombinant secreted therapeuticprotein. The method may further comprise (d) purifying the recombinantsecreted therapeutic protein.

In order to reduce expression of the host cell protein in the method ofthe invention, the gene encoding the host cell protein may comprise agenetic modification that inhibits expression of said host cell protein,or the mammalian cell may comprise a RNA oligonucleotide that inhibitsexpression of the gene encoding said host cell protein byRNA-interference. Reduced expression of the host cell protein means thatthe protein expression of TBC1D20 and CERS2 or the protein expression ofATF6B in the mammalian cell is reduced compared to the same mammaliancell not containing said genetic modification(s) or RNAoligonucleotide(s).

The mammalian cells and the mammalian cells produced by the method ofthe invention may further be used for the production of a recombinantsecreted therapeutic protein or increasing the yield of the recombinantsecreted therapeutic protein. The invention therefore also relates to ause of the mammalian cell of the invention or the mammalian cellproduced by the method of the invention for increasing the yield of arecombinant secreted therapeutic protein. It further relates to a use ofthe mammalian cell of the invention or the mammalian cell produced bythe method of the invention for production of a recombinant secretedtherapeutic protein.

The recombinant secreted therapeutic protein produced by the mammaliancell or the method of the invention is preferably an antibody,preferably a monoclonal antibody, a bi-specific antibody or a fragmentthereof, or a Fc-fusion protein.

Reduced Expression of the Host Cell Proteins TBC1D20, CERS2 or ATF6B

The expression of the host cell proteins TBC1D20, CERS2 or ATF6B isreduced in a mammalian cell according to the invention or used orproduced in the methods of the invention. This means that the proteinexpression of TBC1D20, CERS2 or ATF6B in the mammalian cell is reducedcompared to the same mammalian cell not containing said geneticmodification or RNA oligonucleotide by gene knockdown or gene knockout.

In one embodiment, the expression of the host cell proteins TBC1D20 andCERS2 is reduced. The host cell protein TBC1D20 refers to hamsterTBC1D20 expressed in CHO cells such as encoded by the cDNA sequence ofSEQ ID NO: 1, or having the amino acid sequence of SEQ ID NO: 4, or anyhomologues thereof. As used herein, a homologue thereof means a proteinhaving a sequence identity of at least 80%, at least 85%, at least 90%,at least 95% or at least 98% to the amino acid sequence of SEQ ID NO: 4.The host cell protein CERS2 refers to the hamster CERS2 expressed in CHOcells, such as encoded by the cDNA sequence of SEQ ID NO: 2 or havingthe amino acid sequence of SEQ ID NO: 5 or any homologues thereof. Asused herein, a homologue thereof means a protein having a sequenceidentity of at least 80%, at least 85%, at least 90%, at least 95% or atleast 98% to the amino acid sequence of SEQ ID NO: 5.

In another embodiment, the expression of ATF6B is reduced compared tocontrol cells. The host cell protein ATF6B refers to the hamster ATF6Bexpressed in CHO cells, such as encoded by the cDNA sequence of SEQ IDNO: 3 or having the amino acid sequence of SEQ ID NO: 6 or any homologuethereof. As used herein, a homologue thereof means a protein having asequence identity of at least 80%, at least 85%, at least 90%, at least95% or at least 98% to the amino acid sequence of SEQ ID NO: 6. Theperson skilled in the art would understand that also the expression ofhost cell proteins ATF6B, TBC1D20 and CERS2 may be reduced.

The term “knockdown” or “knockdown technology” refers to a technique ofgene silencing in which the expression of a target gene or gene ofinterest is reduced as compared to the gene expression prior to theintroduction of an RNA oligonucleotide that inhibits expression of atarget gene by RNA-interference, such as by using siRNA or shRNA, whichcan lead to the inhibition of production of the target gene product.“Double knockdown” is the knockdown of two genes, such as the genesencoding for TBC1D20 and CERS2.

In one embodiment, the mammalian cell comprises a RNA oligonucleotidethat inhibits expression of the gene encoding said host cell protein byRNA-interference, wherein host cell protein refers to ATF6B or TBC1D20and CERS2. The skilled person will recognize that the RNAoligonucleotide may be transfected directly into the cell or may beencoded by a polynucleotide sequence within the cell, e.g., by using anexpression vector. Hence, the expression vector comprises apolynucleotide sequence encoding said RNA oligonucleotide. An expressionvector may also comprise a polynucleotide sequence encoding a second RNAoligonucleotide. The two RNA oligonucleotides may be encoded by twoseparate expression cassettes or by the same expression cassetteseparated, e.g., by an IRES sequence. Overexpression of siRNA(s) orshRNA(s) targeting ATF6B or to the combination of TBC1D20 and CERS2,leads to an enhanced production and/or secretion of the secretedrecombinant therapeutic protein in a mammalian expression system.

Preferably the RNA oligonucleotide mediates mRNA repression by completesequence complementarity (i.e., perfect base paring between theantisense strand of the RNA duplex of the small interfering RNA and thetarget mRNA) and is therefore specific for its target. Complete sequencecomplementarity of perfect base paring as used herein means that theantisense strand of the RNA duplex of the small interfering RNA has atleast 89% sequence identity with the target mRNA for at least 15continuous nucleotides, at least 16 continuous nucleotides, at least 17continuous nucleotides, at least 18 continuous nucleotides andpreferably at least 19 continuous nucleotides, or preferably at least93% sequence identity with the target mRNA for at least 15 continuousnucleotides, at least 16 continuous nucleotides, at least 17 continuousnucleotides, at least 18 continuous nucleotides and preferably at least19 continuous nucleotides. Preferably the antisense strand of the RNAduplex of the small interfering RNA has 100% sequence identity with thetarget mRNA for at least 15 continuous nucleotides, at least 16continuous nucleotides, at least 17 continuous nucleotides, at least 18continuous nucleotides and preferably at least 19 continuousnucleotides. The skilled person will understand that miRNAs do notmediate mRNA repression by complete sequence complementarity and aretherefore not gene-specific. Thus, in one embodiment, the RNAoligonucleotide is not a miRNA.

Preferably the RNA-interference is mediated by small hairpin RNA (shRNA)or short interfering RNA (siRNA). The mammalian cell may be transfectedwith one or more expression vector(s) encoding said siRNA(s) orshRNA(s). Preferably the mammalian cell is stably transfected with oneor more expression vector(s) encoding said siRNA(s) or shRNA(s). The RNAoligonucleotide may be constitutively expressed or conditionallyexpressed. For example, expression of the RNA oligonucleotide may besilent during growth phase and switched on during protein productionphase.

An exemplary siRNA for knocking-down TBC1D20 is siTbc1D20 #1 (SEQ ID NO:7). An exemplary siRNA for knocking-down CERS2 is siCerS2 #1 (SEQ ID NO:8). Preferably the siRNAs having the sequence of SEQ ID NOs: 7 or 8 areused in CHO cells. These siRNAs can be used independently of each other.Thus, each of siTbc1D20 #1 or siCERS2 #1 may be used or both siRNAs maybe used. While the expression of both, TBC1D20 and CERS2, is reducedaccording to the method or the mammalian cell of the invention, themeans to achieve the reduction of the host cell proteins are independentof each other. Thus, expression of host cell protein TBC1D20 may bereduced by gene knockdown using siRNA and expression of host cellprotein CERS2 may be reduced by gene knockdown using shRNA, or viceversa. Alternatively, the expression of host cell protein TBC1D20 may bereduced by gene knockdown, e.g., using siRNA and expression of host cellprotein CERS2 may be reduced by gene knockout, or vice versa.

An exemplary shRNA for knocking-down TBC1D20 comprises shTbc1D20 #1 (SEQID NO: 12); an exemplary shRNA for knocking-down CERS2 comprises shCerS2#1 (SEQ ID NO: 13) or shCerS2 #2 (SEQ ID NO: 14), or a combination ofsaid shRNAs. Preferably, the shRNAs comprising the sequence of SEQ IDNOs: 12, 13 or 14, are used in CHO cells. Exemplary DNA oligonucleotidesencoding shRNAs suitable in the present invention for knocking-downTBC1D20 (SEQ ID NOs: 16 and 17) or CERS2 (SEQ ID NOs 18-21) are shownbelow. Preferably the DNA oligonucleotide encoding the shRNA targetingTBCD1 D20 comprises the sequence of nucleotides 6 to 26 of SEQ ID NO: 16(shTBC1D20 #1 oligonucleotide forward) and more preferably the sequenceof nucleotides 6 to 26 and 46 to 64 of SEQ ID NO: 16 (shTBC1D20 #1oligonucleotide forward). Preferably the DNA oligonucleotide encodingthe shRNA targeting CERS2 comprises the sequence of nucleotides 6 to 26of SEQ ID NOs: 18 or 20 (shCERS2 #1 or #2 oligonucleotide forward) andmore preferably the sequence of nucleotides 6 to 26 and 46 to 64 of SEQID NOs: 18 or 20 (shCERS2 #1 or #2 oligonucleotide forward).

These shRNAs can be used independently of each other. Thus, shTbc1D20#1, shCERS2 #1 or shCERS2 #2 may be used, or shTbc1D20 #1 and shCERS2 #1or shCERS2 #2 may be used. While the expression of both, TBC1D20 andCERS2 is reduced according to the method or the mammalian cell of theinvention, the means to achieve the reduction of the host cell proteinsare independent of each other. Thus, expression of host cell proteinTBC1D20 may be reduced by gene knockdown using siRNA and expression ofhost cell protein CERS2 may be reduced by gene knockdown using shRNA, orvice versa. Alternatively, the expression of host cell protein TBC1D20may be reduced by gene knockdown, e.g., using shRNA, and the expressionof host cell protein CERS2 may be reduced by gene knockout, or viceversa.

Exemplary siRNAs for knocking-down ATF6B are siAtf6b #1 (SEQ ID NO: 9),siAtf6b #2 (SEQ ID NO: 10), or siAtf6b #3 (SEQ ID NO: 11). Preferablyone or more of siAtf6b #1 (SEQ ID NO: 9), siAtf6b #2 (SEQ ID NO: 10),and siAtf6b #3 (SEQ ID NO: 11) are used according to the invention, morepreferably one or more of siAtf6b #1 (SEQ ID NO: 9) and siAtf6b #2 (SEQID NO: 10) are used. An exemplary shRNA for knocking-down ATF6Bcomprises shAtf6b #1 (SEQ ID NO: 15) or shAtf6b #2 (SEQ ID NO: 37),preferably shAtf6b #1 (SEQ ID NO: 15). Preferably the siRNAs having thesequence of SEQ ID NOs: 9, 10 or 11 or the shRNA comprising the sequenceof SEQ ID NOs: 15 or 37 are used in CHO cells. Exemplary DNAoligonucleotides encoding shRNAs suitable in the present invention forknocking-down ATF6B are shown below (SEQ ID NOs: 22, 23, 35 and 36).Preferably the DNA oligonucleotide encoding the respective shRNAtargeting ATF6B comprises the sequence of nucleotides 6 to 26 of SEQ IDNOs: 22 or 35 (shATF6B #1 or #2 oligonucleotide forward) and morepreferably the sequence of nucleotides 6 to 26 and 46 to 64 of SEQ IDNOs: 22 or 35 (shATF6B #1 or #2 oligonucleotide forward).

In another embodiment, at least one gene encoding a host cell proteincomprises a genetic modification that inhibits expression of said hostcell protein. The genetic modification in the gene(s) encoding the hostcell protein(s) TBC1D20, CERS2 or ATF6B may be independent of each othera gene deletion or a mutation in the gene that inhibits expression ofthe host cell protein. The mutation may be a deletion, addition orsubstitution. The skilled person would know that the mutation may be inthe coding region of the gene and/or the mutation may be in the promoteror a regulatory region of the gene as long as the gene expression isreduced. Preferably, the mutation is in the promoter or regulatoryregion of the gene. The mutation may be introduced using methods knownin the art. The skilled person would understand that the same effect maybe achieved using overexpression of a dominant mutant host cell proteinthat has reduced protein activity. Alternatively, the host cell proteingene may be deleted in the mammalian cell and an expression cassetteencoding said host cell protein under the control of a weak promoter maybe introduced into the mammalian cell, resulting in an overall reducedexpression of the host cell protein compared to a control cell (i.e.,the same mammalian cell not containing said gene deletion). This genemutation may be either in one or both alleles of a gene.

Reduced host cell protein expression can be determined by comparing theprotein expression of TBC1D20, CERS2 or ATF6B in the mammalian cellcompared to a control mammalian cell, i.e., the same mammalian cell notcontaining said genetic modification or RNA oligonucleotide. Preferably,the expression of the host cell protein ATF6B or of host cell proteinsTBC1D20, CERS2 are reduced by at least 30%, at least 40%, at least 50%,at least 75%, or 100%, compared to a control mammalian cell. This may bemeasured on protein level, e.g., by ELISA, by Western blotting, byradioimmunoassays, immunoprecipitation, assaying for the biologicalactivity of the protein, by immunostaining of the protein followed byFACS analysis or by homogeneous time-resolved fluorescence (HTRF)assays, or any other suitable method known in the art for quantifyingprotein. The reduced expression of host cell protein ATF6Bor of hostcell proteins TBC1D20, CERS2 may also be determined on mRNA level, e.g.,by quantitative PCR or any other suitable method known in the art forquantifying mRNA. mRNA transcribed from a selected sequence can furtherbe quantified by Northern blot hybridization, ribonuclease RNAprotection, in situ hybridization to cellular RNA or by PCR.

It is particularly important that the host cell protein expression isreduced during production phase. It is therefore possible that knockdownof host cell protein expression induced after growth phase or early inproduction phase as long as host cell protein expression is reducedduring most of the production phase. Host cell protein expression shouldbe reduced at least 3 days before end of culture, at least 5 days beforeend of culture, at least 7 days before end of culture or at least 9 daysbefore end of culture. Preferably the host cell protein expression isreduced throughout cell culture.

A gene may also be modified by deleting the gene using “knockout”technology. The term “knockout” refers to cells which have beengenetically modified so that the expression of host cell proteins AFT6Bor the combination of host cell proteins TBC1D20 and CERS2 is/areinhibited and the respective host cell protein is not produced(reduction by 100%). This may be achieved using various technologieswhich are known in the art to the skilled person, including CRISPR-Cas9or Zinc finger nuclease technology. Alternatively, the gene may bealtered to inhibit the expression of its protein by introduction of amutation in the gene. The gene mutation may be a nucleotide deletion,addition or substitution in the coding region or in the promoter orregulatory region of the gene. This gene mutation may be either in oneor both alleles of a gene.

Enhanced Secretion of the Recombinant Therapeutic Protein

The reduced expression of ATF6B or TBC1D20 and CERS2 in a mammalian cellaccording to the invention results in an enhanced secretion of therecombinant therapeutic protein. Protein secretion can be increased byimproved cell density or cell viability. It may also be increased byimproved specific cell productivity. However, the skilled person willunderstand that having improved cell density or cell viability onlyenhances the total yield of the secreted recombinant therapeutic proteinin case the specific cell productivity is not substantially affected oreven improved. Likewise having increased specific cell productivity onlyenhances the total yield of the secreted recombinant therapeutic proteinin case the cell density or cell viability is not substantially affectedor even improved. Enhanced secretion of the recombinant therapeuticprotein therefore refers to the total yield of the recombinanttherapeutic protein in the cell culture, typically measured as aconcentration (titer), such as mg/ml. The secretion of the recombinanttherapeutic protein according to the invention is enhanced by at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least75%, at least 100% or at least 200%, compared to a control mammaliancell, i.e., not containing said genetic modification or RNAoligonucleotide. In a preferred embodiment yield of the secretion of therecombinant therapeutic protein is enhanced at harvest.

The protein ATF6b is involved in unfolded protein responses (UPR). Ithas been described before that only “ATF6α, but neither ATF6β nor ATF4,has the ability to trigger expansion of the ER” (Bommiasamy et al.,2009, Journal of Cell Sciences. 122: 1626-1636) and that only ATF6a isresponsible for transcriptional induction of ER chaperones (Yamamoto etal., 2007, Cell. 13: 365-376). It was further reported that ATF6b is avery weak transcriptional activator of ER stress response (ERSR) genescompared to ATF6a (Thuerauf et al., 2007, The Journal of BiologicalChemistry. 282(31): 22865-22878). Without being bound by theory, wetherefore believe that depletion of ATF6b in producer cell lines mightaugment the transcriptional activity of ATF6a and thus increase theprotein folding capacity of the ER by triggering the expression of ERchaperones.

Ceramides of varying chain lengths are synthesized by six differentisoforms of Ceramide Synthases (CERS1-6) in the ER. CERS2 catalyzes thesynthesis of very long chain ceramides (C20-C26) and its depletion inmice has been shown to decrease very long chain ceramides (>022) whilstinducing a compensatory increase in C16-C18 ceramides (Grösch S, et al.,2012. Progress in Lipid Research. 51:50-62). CERT efficiently transfersceramides having C14, C16, C18, and C20 chains, but it does not transferlonger acyl chains (Kumagai D, et al., 2005. The Journal of BiologicalChemistry. 280(8):6488-6495). Thus, without being bound by theory, theefficiency of the CERT mediated ceramide transport might be increased bydepleting CERS2, resulting in a higher amount of proteins that aretransferred to the plasma membrane.

The small GTPase Rab1 has a crucial role in the vesicular transport ofproteins that have been processed in the ER. GTP-bound Rab1 is requiredto maintain the Golgi Apparatus (Haas A, et al., 2007. Journal of CellScience. 120:2997-3010). TBC1D20 is a GTPase-activating protein (GAP).It catalyzes the conversion of active, GTP-bound Rab1 into an inactive,GDP-bound state. Thus, without being bound by theory, depletion ofTBC1D20 might result in a constitutive active form of Rab1, positivelyaffecting vesicular protein transport process.

TBC1D20 and CERS2 are both involved in protein transport at the Golgiapparatus. Reduction of TBC1D20 and CERS2 expression therefore seem toact synergistically, resulting in an increased recombinant proteinsecretion compared to reduction of each of said proteins alone.

Secreted Recombinant Therapeutic Protein

The secreted recombinant therapeutic protein produced in the mammaliancells of the invention includes, but is not limited to an antibodies ora fusion protein, such as a Fc-fusion proteins. Other secretedrecombinant therapeutic proteins can be for example enzymes, cytokines,lymphokines, adhesion molecules, receptors and derivatives or fragmentsthereof, and any other polypeptides and scaffolds that can serve asagonists or antagonists and/or have therapeutic or diagnostic use.

Other recombinant proteins of interest are for example, but not limitedto insulin, insulin-like growth factor, hGH, tPA, cytokines, such asinterleukins (IL), e.g. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega or IFN tau, tumornecrosis factor (TNF), such as TNF alpha and TNF beta, TNF gamma, TRAIL;G-CSF, GM-CSF, M-CSF, MCP-1, and VEGF. Also included is the productionof erythropoietin or any other hormone growth factors and any otherpolypeptides that can serve as agonists or antagonists and/or havetherapeutic or diagnostic use.

A preferred recombinant secreted therapeutic protein is an antibody or afragment or derivative thereof. Thus, the invention can beadvantageously used for production of antibodies such as monoclonalantibodies, multispecific antibodies, or fragments thereof, preferablyof monoclonal antibodies, bi-specific antibodies or fragments thereof.Furthermore, the method for producing a recombinant secreted therapeuticprotein according to the invention can be advantageously used forproduction of antibodies such as monoclonal antibodies, multispecificantibodies, or fragments thereof, preferably of monoclonal antibodies,bi-specific antibodies or fragments thereof. Exemplary antibodies withinthe scope of the present invention include but are not limited toanti-CD2, anti-CD3, anti-CD20, anti-CD22, anti-CD30, anti-CD33,anti-CD37, anti-CD40, anti-CD44, anti-CD44v6, anti-CD49d, anti-CD52,anti-EGFR1 (HER1), anti-EGFR2 (HER2), anti-GD3, anti-IGF, anti-VEGF,anti-TNFalpha, anti-IL2, anti-IL-5R or anti-IgE antibodies, and arepreferably selected from the group consisting of anti-CD20, anti-CD33,anti-CD37, anti-CD40, anti-CD44, anti-CD52, anti-HER2/neu (erbB2),anti-EGFR, anti-IGF, anti-VEGF, anti-TNFalpha, anti-IL2 and anti-IgEantibodies.

Antibody fragments include e.g. “Fab fragments” (Fragmentantigen-binding=Fab). Fab fragments consist of the variable regions ofboth chains, which are held together by the adjacent constant region.These may be formed by protease digestion, e.g. with papain, fromconventional antibodies, but similar Fab fragments may also be producedby genetic engineering. Further antibody fragments include F(ab′)2fragments, which may be prepared by proteolytic cleavage with pepsin.

Using genetic engineering methods it is possible to produce shortenedantibody fragments which consist only of the variable regions of theheavy (VH) and of the light chain (VL). These are referred to as Fvfragments (Fragment variable=fragment of the variable part). Since theseFv-fragments lack the covalent bonding of the two chains by thecysteines of the constant chains, the Fv fragments are often stabilized.It is advantageous to link the variable regions of the heavy and of thelight chain by a short peptide fragment, e.g. of 10 to 30 amino acids,preferably 15 amino acids. In this way a single peptide strand isobtained consisting of VH and VL, linked by a peptide linker. Anantibody protein of this kind is known as a single-chain-Fv (scFv).Examples of scFv-antibody proteins are known to the person skilled inthe art.

In recent years, various strategies have been developed for preparingscFv as a multimeric derivative. This is intended to lead, inparticular, to recombinant antibodies with improved pharmacokinetic andbiodistribution properties as well as with increased binding avidity. Inorder to achieve multimerisation of scFv, scFv were prepared as fusionproteins with multimerisation domains. The multimerisation domains maybe, e.g. the CH3 region of an IgG or coiled coil structure (helixstructures) such as Leucin-zipper domains. However, there are alsostrategies in which the interactions between the VHNL regions of thescFv are used for the multimerisation (e.g. dia-, tri- and pentabodies).By diabody the skilled person means a bivalent homodimeric scFvderivative. The shortening of the Linker in a scFv molecule to 5-10amino acids leads to the formation of homodimers in which an inter-chainVH/VL-superimposition takes place. Diabodies may additionally bestabilized by the incorporation of disulphide bridges. Examples ofdiabody-antibody proteins are known to the person skilled in the art.

Preferred secreted recombinant therapeutic antibodies according to theinvention are bispecific antibodies. Bispecific antibodies typicallycombine antigen-binding specificities for target cells (e.g., malignantB cells) and effector cells (e.g., T cells, NK cells or macrophages) inone molecule. Exemplary bispecific antibodies, without being limitedthereto are diabodies, BiTE (Bi-specific T-cell Engager) formats andDART (Dual-Affinity Re-Targeting) formats. The diabody format separatescognate variable domains of heavy and light chains of the two antigenbinding specificities on two separate polypeptide chains, with the twopolypeptide chains being associated noncovalently. The DART format isbased on the diabody format, but it provides additional stabilizationthrough a C-terminal disulfide bridge.

By triabody the skilled person means a trivalent homotrimeric scFvderivative. In said scFv derivatives the VH-VL domains are fuseddirectly without a linker sequence, which leads to the formation oftrimers. The skilled person will also be familiar with so-calledminiantibodies which have a bi-, tri- or tetravalent structure and arederived from scFv. The multimerisation is carried out by di-, tri- ortetrameric coiled coil structures.

Also anticipated in the context of the present invention are minibodies.By minibody, the skilled person means a bivalent, homodimeric scFvderivative. It consists of a fusion protein which contains the CH3region of an immunoglobulin, preferably IgG, most preferably IgG1 as thedimerisation region which is connected to the scFv via a Hinge region(e.g. also from IgG1) and a Linker region. Examples of minibody-antibodyproteins are known to the person skilled in the art.

Another preferred recombinant secreted therapeutic protein is a fusionprotein, such as Fc-fusion protein. Thus, the invention can beadvantageously used for production of fusion proteins, such as Fc-fusionproteins. Furthermore, the method for producing a secreted recombinanttherapeutic protein according to the invention can be advantageouslyused for production of fusion proteins, such as Fc-fusion proteins.

The effector part of the fusion protein can be the complete sequence orany part of the sequence of a natural or modified heterologous proteinor a composition of complete sequences or any part of the sequence of anatural or modified heterologous protein. The immunoglobulin constantdomain sequences may be obtained from any immunoglobulin subtypes, suchas IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2 subtypes or classes such as IgA,IgE, IgD or IgM. Preferentially they are derived from humanimmunoglobulin, more preferred from human IgG and even more preferredfrom human IgG1 and IgG3. Non-limiting examples of Fc-fusion proteinsare MCP1-Fc, ICAM-Fc, EPO-Fc and scFv fragments or the like coupled tothe CH2 domain of the heavy chain immunoglobulin constant regioncomprising the N-linked glycosylation site. Fc-fusion proteins can beconstructed by genetic engineering approaches by introducing the CH2domain of the heavy chain immunoglobulin constant region comprising theN-linked glycosylation site into another expression construct comprisingfor example other immunoglobulin domains, enzymatically active proteinportions, or effector domains. Thus, an Fc-fusion protein according tothe present invention comprises also a single chain Fv fragment linkedto the CH2 domain of the heavy chain immunoglobulin constant regioncomprising e.g. the N-linked glycosylation site.

The recombinant secreted therapeutic protein, especially the antibody,antibody fragment or Fc-fusion protein is preferably recovered/isolatedfrom the culture medium as a secreted polypeptide. It is necessary topurify the recombinant secreted therapeutic protein from otherrecombinant proteins and host cell proteins to obtain substantiallyhomogenous preparations of the recombinant secreted therapeutic protein.As a first step, cells and/or particulate cell debris are removed fromthe culture medium or lysate. Further, the recombinant secretedtherapeutic protein is purified from contaminant soluble proteins,polypeptides and nucleic acids, for example, by fractionation onimmunoaffinity or ion-exchange columns, ethanol precipitation, reversephase HPLC, Sephadex chromatography, and chromatography on silica or ona cation exchange resin such as DEAE. Methods for purifying aheterologous protein expressed by host cells are known in the art.

In one embodiment the recombinant secreted therapeutic protein isencoded by one or more expression cassette(s) encoding the secreted.

In some embodiments, the secreted therapeutic protein may be placedunder the control of an amplifiable genetic selection marker, such asdihydrofolate reductase (DHFR), glutamine synthetase (GS). Theamplifiable selection marker gene can be on the same expression vectoras the secreted therapeutic protein expression cassette. Alternatively,the amplifiable selection marker gene and the secreted therapeuticprotein expression cassette can be on different expression vectors, butintegrate in close proximity into the host cell's genome. Two or morevectors that are co-transfected simultaneously, for example, oftenintegrate in close proximity into the host cell's genome. Amplificationof the genetic region containing the secreted therapeutic proteinexpression cassette is then mediated by adding the amplification agent(e.g., MTX for DHFR or MSX for GS) into the cultivation medium.

Sufficiently high stable levels of the secreted therapeutic protein inthe host cell or the producer cell may also be achieved, e.g., bycloning multiple copies of the secreted therapeutic proteinencoding-polynucleotide into an expression vector. Cloning multiplecopies of the secreted therapeutic protein-encoding polynucleotide intoan expression vector and amplifying the secreted therapeutic proteinexpression cassette as described above may further be combined.

Antibody Production

For producing a recombinant antibody, the DNA molecules encodingfull-length light and heavy chains or fragments thereof are insertedinto an expression vector such that the sequences are operatively linkedto transcriptional and translational control sequences. Alternatively,DNA molecules encoding light chain variable regions and heavy chainvariable regions can be chemically synthesized using the sequenceinformation provided herein. Synthetic DNA molecules can be ligated toother appropriate nucleotide sequences, including, e.g., constant regioncoding sequences, and expression control sequences, to produceconventional gene expression constructs encoding the desired antibody.For manufacturing the antibodies of the invention, the skilled artisanmay choose from a great variety of expression systems well known in theart, e.g. those reviewed by Kipriyanow and Le Gall, 2004. MolecularBiotechnology. 26:39-60. Expression vectors include plasmids,retroviruses, cosmids, EBV-derived episomes, and the like. The term“expression vector” comprises any vector suitable for the expression ofa foreign DNA. Examples of such expression vectors are viral vectors,such as adenovirus, vaccinia virus, baculovirus and adeno-associatedvirus vectors. In this connection, the expression “virus vector” isunderstood to mean both a DNA and a viral particle. Examples of phage orcosmid vectors include pWE15, M13, λEMBL3, λEMBL4, λFIXII, λDASHII,λZAPII, λgT10, λgt11, Charon4A and Charon21A. Examples of plasmidvectors include pBR, pUC, pBluescriptII, pGEM, pTZ and pET groups.Various shuttle vectors may be used, e.g., vectors which mayautonomously replicate in a plurality of host microorganisms such as E.coli and Pseudomonas sp. In addition, artificial chromosome vectors areconsidered as expression vectors. The expression vector and expressioncontrol sequences are selected to be compatible with the host cell.Examples of mammalian expression vectors include, but are not limitedto, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2, pDisplay,pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRepS, D H26S, D HBB, pNMT1,pNMT41, pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which areavailable from Stratagene, pTRES which is available from Clontech, andtheir derivatives.

The antibody light chain gene and the antibody heavy chain gene can beinserted into separate vectors. In certain embodiments, both DNAsequences are inserted into the same expression vector. Convenientvectors are those that encode a functionally complete human CH or CLimmunoglobulin sequence, with appropriate restriction sites engineeredso that any VH or VL sequence can be easily inserted and expressed, asdescribed above, wherein the CH1 and/or upper hinge region comprises atleast one amino acid modification of the invention. The constant chainis usually kappa or lambda for the antibody light chain. The recombinantexpression vector may also encode a signal peptide that facilitatessecretion of the antibody chain from a host cell. The DNA encoding theantibody chain may be cloned into the vector such that the signalpeptide is linked in-frame to the amino terminus of the mature antibodychain DNA. The signal peptide may be an immunoglobulin signal peptide ora heterologous peptide from a non-immunoglobulin protein. Alternatively,the DNA sequence encoding the antibody chain may already contain asignal peptide sequence. In addition to the DNA sequences encoding theantibody chains, the recombinant expression vectors carry regulatorysequences including promoters, enhancers, termination andpolyadenylation signals and other expression control elements thatcontrol the expression of the antibody chains in a host cell. Examplesfor promoter sequences (exemplified for expression in mammalian cells)are promoters and/or enhancers derived from (CMV) (such as the CMVSimian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus,(e. g., the adenovirus major late promoter (AdMLP)), polyoma and strongmammalian promoters such as native immunoglobulin and actin promoters.Examples for polyadenylation signals are BGH polyA, SV40 late or earlypolyA; alternatively, 3′UTRs of immunoglobulin genes etc. can be used.

The recombinant expression vectors may also carry sequences thatregulate replication of the vector in host cells (e.g. origins ofreplication) and selectable marker genes. Nucleic acid moleculesencoding the heavy chain or an antigen-binding portion thereof and/orthe light chain or an antigen-binding portion thereof of an antibody ofthe present invention, and vectors comprising these DNA molecules can beintroduced into host cells, e.g. bacterial cells or higher eukaryoticcells, e.g. mammalian cells, according to transfection methods wellknown in the art, including liposome-mediated transfection,polycation-mediated transfection, protoplast fusion, microinjections,calcium phosphate precipitation, electroporation or transfer by viralvectors.

It is within ordinary skill in the art to express the heavy chain andthe light chain from a single expression vector or from two separateexpression vectors. Preferably, the DNA molecules encoding the heavychain and the light chain are present on two vectors which areco-transfected into the host cell, preferably a mammalian cell.

Mammalian cell lines available as hosts for expression are well known inthe art and include, inter alia, Chinese hamster ovary (CHO, CHO-DG44,CHO-K1) cells, NSO, SP2/0 cells, HeLa cells, HEK293 cells, baby hamsterkidney (BHK) cells, monkey kidney cells (COS), human carcinoma cells (e.g., HepG2), A549 cells, 3T3 cells or the derivatives/progenies of anysuch cell line. Other mammalian cells, including but not limited tohuman, mice, rat, monkey and rodent cells lines, or other eukaryoticcells, including but not limited to yeast, insect and plant cells, orprokaryotic cells such as bacteria may be used. The antibody moleculesof the invention are produced by culturing the host cells for a periodof time sufficient to allow for expression of the antibody molecule inthe host cells. Following expression, the intact antibody (or theantigen-binding fragment of the antibody) can be harvested and purifiedusing techniques well known in the art, e.g., Protein A, Protein G,affinity tags such as glutathione-S-transferase (GST) and histidinetags.

Protein Purification

The recombinant secreted therapeutic proteins are preferably recoveredfrom the culture medium as a secreted polypeptide. It is necessary topurify the recombinant secreted therapeutic proteins using standardprotein purification methods used for recombinant proteins in a way thatsubstantially homogenous preparations of the protein are obtained. Byway of example, state-of-the art purification methods useful forobtaining the recombinant secreted therapeutic protein of the inventioninclude, as a first step, removal of cells and/or particulate celldebris from the culture medium or lysate. The recombinant secretedtherapeutic protein is then purified from contaminant soluble proteins,polypeptides and nucleic acids, for example, by fractionation onimmunoaffinity or ion-exchange columns, ethanol precipitation, reversephase HPLC, Sephadex chromatography, chromatography on silica or on acation exchange resin. Antibodies or Fc-fusion proteins, e.g., may bepurified by standard protein A chromatography, e.g., using protein Aspin columns (GE Healthcare). Protein purity may be verified by reducingSDS PAGE. recombinant secreted therapeutic protein concentrations may bedetermined by measuring absorbance at 280 nm and utilizing the proteinspecific extinction coefficient. As a final step in the process forobtaining an recombinant secreted therapeutic protein preparation, thepurified recombinant secreted therapeutic protein may be dried, e.g.lyophilized, as described below for therapeutic applications.

Pharmaceutical Compositions

To be used in therapy, the recombinant secreted therapeutic protein maybe formulated into a pharmaceutical composition appropriate tofacilitate administration to animals or humans. Pharmaceuticalcompositions containing the recombinant secreted therapeutic protein canbe presented in a dosage unit form and can be prepared by any suitablemethod. Typical formulations of a recombinant secreted therapeuticprotein can be prepared by mixing the protein with physiologicallyacceptable carriers, excipients or stabilizers, in the form oflyophilized or otherwise dried formulations or aqueous solutions oraqueous or non-aqueous suspensions. Carriers, excipients, modifiers orstabilizers are nontoxic at the dosages and concentrations employed.They include buffer systems such as phosphate, citrate, acetate andother anorganic or organic acids and their salts; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone or polyethylene glycol (PEG); amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, oligosaccharides or polysaccharides andother carbohydrates including glucose, mannose, sucrose, trehalose,dextrins or dextrans; chelating agents such as EDTA; sugar alcohols suchas, mannitol or sorbitol; salt-forming counter-ions such as sodium;metal complexes (e.g. Zn-protein complexes); and/or ionic or non-ionicsurfactants such as TWEEN™ (polysorbates), PLURONICS™ or fatty acidesters, fatty acid ethers or sugar esters. Also organic solvents can becontained in the recombinant secreted therapeutic protein formulationsuch as ethanol or isopropanol. The excipients may also have arelease-modifying or absorption-modifying function.

The recombinant secreted therapeutic protein may also be dried(freeze-dried, spray-dried, spray-freeze dried, dried by near orsupercritical gases, vacuum dried, air-dried), precipitated orcrystallized or entrapped in microcapsules that are prepared, forexample, by coacervation techniques or by interfacial polymerizationusing, for example, hydroxymethylcellulose or gelatin andpoly-(methylmethacylate), respectively, in colloidal drug deliverysystems (for example, liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules), in macroemulsions or precipitated orimmobilized onto carriers or surfaces, for example by pcmc technology(protein coated microcrystals). Such techniques are disclosed inRemington: The Science and Practice of Pharmacy, 21st edition,Hendrickson R. Ed.

Naturally, the formulations to be used for in vivo administration mustbe sterile; sterilization may be accomplished be conventionaltechniques, e.g. by filtration through sterile filtration membranes. Itmay be useful to increase the concentration of the antibody to come to aso-called high concentration liquid formulation (HCLF); various ways togenerate such HCLFs have been described.

The recombinant secreted therapeutic protein may also be contained in asustained-release preparation. Further, the recombinant secretedtherapeutic protein can be incorporated in other application forms, suchas dispersions, suspensions or liposomes, tablets, capsules, powders,sprays, transdermal or intradermal patches or creams with or withoutpermeation enhancing devices, wafers, nasal, buccal or pulmonaryformulations, or may be produced by implanted cells or—after genetherapy—by the individual's own cells.

A recombinant secreted therapeutic protein may also be derivatized witha chemical group such as polyethylene glycol (PEG), a methyl or ethylgroup, or a carbohydrate group. These groups may be useful to improvethe biological characteristics of the antibody, e.g. to increase serumhalf-life or to increase tissue binding.

The preferred mode of application is parenteral, by infusion orinjection (intravenous, intramuscular, subcutaneous, intraperitoneal,intradermal), but other modes of application such as by inhalation,transdermal, intranasal, buccal, oral, may also be applicable.

For the prevention or treatment of disease, the appropriate dosage ofthe recombinant secreted therapeutic protein will depend on the type ofdisease to be treated, the severity and course of the disease, whetherthe recombinant secreted therapeutic protein is administered forpreventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the recombinant secreted therapeuticprotein, and the discretion of the attending physician. The recombinantsecreted therapeutic protein is suitably administered to the patient atone time or over a series of treatments.

Materials and Methods

TABLE 3 Cell lines use in the Examples Designation Description SpeciesCHO-mAb1 CHO-DG44-based producer cell clone Hamster secreting the IgG1antibody mAb1 (with a heavy chain with a sequence according to SEQ IDNO: 24 and a light chain sequence according to SEQ ID NO: 25) CHO-mAb2CHO-DG44-based producer cell clone Hamster secreting the IgG1 antibodymAb2 (with a heavy chain sequence according to SEQ ID NO: 26 and a lightchain sequence according to SEQ ID NO: 27)

Cell Culture of Suspension Cells

Suspension cultures of mAb producing CHO-DG44 cells (Urlaub G, et al.,1986. Somatic Cell and Molecular Genetics. 12(6):555-566) and stabletransfectants thereof were incubated in a chemically defined, serum-freemedium. Seed stock cultures were sub-cultivated every 2-3 days withseeding densities of 3×10⁵-2×10⁵ cells/mL, respectively. Cells weregrown in T-flask (Greiner). T-flasks were incubated in humidifiedincubators (Varolab) at 37° C. and 5% CO₂. The cell concentration andviability was determined by trypan blue exclusion using a countingchamber.

Fed-Batch Cultivation

Cells were seeded at 3×10⁵ cells/ml into 125 ml shake flasks (Corning)in 30 ml of chemically defined, serum-free medium without antibiotics orMTX. The cultures were agitated at 120 rpm in 37° C. and 5% CO₂ in aminitron incubator (Infors) which was reduced to 2% following day 3.Feed solution was added daily from day 3 on and glucose was measuredusing the offline glucose analysis device “LaboTRACE” (Trace Analytics).At concentrations below 3 g/L, glucose (#G8769, Sigma-Aldrich) wasadjusted to 5 g/L. Cell densities and viability were determined bytrypan-blue exclusion using the “Countess 2 FL automated cell counter”(Life Technologies). Cumulative specific productivity was calculated asproduct concentration analyzed by ELISA at the given day divided by the“integral of viable cells” (IVC) until that time point.

Generation of Antibody-Producing Cells

CHO-DG44 cells (Urlaub G, et al., 1983. Cell. 33:405-412) were stablytransfected with expression plasmids encoding the IgG1 antibody mAb1(with a heavy chain with a sequence according to SEQ ID NO: 24 and alight chain sequence according to SEQ ID NO: 25) and are referred to asCHO-mAb1 herein. Selection was carried out by cultivation of transfectedcells in the absence of Hypoxanthine and Thymidine and in the presenceof the respective selective agents, for which a resistance cassette isencoded by the expression plasmids. After about 3 weeks of selection,stable cell populations are obtained and further cultivated according toa standard stock culture regime with subcultivation every 2 to 3 days.Increasing concentrations of methotrexate were added step-wise toincrease mAb1 gene expression. In a next step, FACS-based single cellcloning of the stably transfected cell populations was carried out togenerate monoclonal cell lines.

CHO-DG44 cells (Urlaub G, et al., 1983. Cell. 33:405-412) werealternatively stably transfected with expression plasmids encoding theIgG1 antibody mAb2 (with a heavy chain with a sequence according to SEQID NO: 26 and a light chain sequence according to SEQ ID NO: 27) and arereferred to as CHO-mAb2 herein. Selection was carried out by cultivationof transfected cells in the absence of hypoxanthine and thymidine and inthe in the presence of the respective selective agents, for which aresistance cassette is encoded by the expression plasmids. After about 3weeks of selection, stable cell populations were obtained and furthercultivated according to a standard stock culture regime withsubcultivation every 2 to 3 days. Increasing concentrations ofmethotrexate were added step-wise to increase mAb2 gene expression. In anext step, ClonePixFL-based single cell cloning of the stablytransfected cell populations was carried out to generate monoclonal celllines.

Stably transfected CHO-mAb1 or CHO-mAb2 cells were cultivated inchemically defined, serum-free medium (Boehringer-Ingelheim)supplemented with G418 (Gibco, Life technologies). CHO-mAb1 cell mediumwas supplemented with 400 nM MTX and CHO-mAb2 cell medium wassupplemented with 100 nM MTX (Sigma-Aldrich, Germany). Cells weresubcultivated every 2 or 3 days with a seeding density of 3×10⁵ cells/mLor 2×10⁵ cells/mL.

Transient Expression of Human microRNAs in CHO Producer Cells

CHO-DG44 cells stably secreting an IgG1 antibody (mAb1) were cultivatedin chemically defined, serum-free medium (Boehringer-Ingelheim)supplemented with G418 (Gibco, Life technologies) and 400 nM MTX(Sigma-Aldrich, Germany) and were subcultivated every 2 or 3 days with aseeding density of 3×10⁵ cells/mL or 2×10⁵ cells/mL.

Cells were transfected via nucleofection one day after subcultivation(4×10⁵ cells/sample) in 96-well Nucleofector kit SG (Lonza) containing 1μM miRNA using the Amaxa 96-well Shuttle Device (Lonza) and program96-DT-133 according to the manufacturer's instructions. For transienttransfection a negative control miRNA miR-c #1 (miRIDIAN microRNA mimicnegative control #1, Dharmacon) was used. Cells were then seeded with adensity of 3×10⁵ cells/mL into a 24-well flat bottom plate (Greiner).One day after transfection the volume of the medium was doubled byaddition of fresh medium. Two days after transfection total RNA wasextracted using the RNeasy Plus Mini Kit (Qiagen) and mRNA levels oftarget genes were determined by qPCR as described below.

Next Generation Sequencing

CHO-DG44 cells stably secreting an IgG1 antibody (mAb1) were transfectedvia nucleofection one day after subcultivation (4×10⁵ cells/sample) in96-well Nucleofector kit SG (Lonza) containing 1 μM miRNA (miR-c #1,hsa-miR-1287 or hsa-miR-1978) using the Amaxa 96-well Shuttle Device(Lonza) and program 96-DT-133 as described above. 12 hours aftertransfection total RNA was extracted using RNeasy Plus Mini Kit (Qiagen)according to manufacturer's instructions and quality and quantity wereanalyzed with the 2100 Bioanalyzer (Agilent) using RNA 6000 Nano Kit.Subsequently, cDNA libraries were generated with 200 ng RNA using TruSeqRNA Sample Prep Kit v2 (Illumina) according to the manufacturer'sinstructions. Briefly, polyA+RNA was enriched, followed by randomfragmentation of poly-A+RNA (100-500 bp), synthesis of ds cDNA withoutstrand specificity, ligation of barcode-labeled adapters per sample andamplification of asymmetrically ligated fragments. Quality and quantitywas analyzed with a 2100 Bioanalyzer (Agilent) using DNA 1000 Kit. ThecDNA library was loaded with 9 pmol per lane on a full flow cell on aHiSeq 2000 sequencing instrument (Illumina) with single-read mode and 60cycles of fragment sequencing plus 7 cycles of barcode sequencing.Sequence reads per sample were mapped with TopHat software against thereference genome from Cricetulus griseus (assembly “CriGri_1.0”https://www.ncbi.nlm.nih.gov/assembly/309608). Quantification wascarried out according to Mortazavi et al. (Nature Methods—5, 621-628(2008)). Thus, transcript abundance is calculated as RPKM (=reads perkilobase of exon model per million mapped reads).

Transient Expression of Human siRNAs in CHO Producer Cells

CHO-mAb1 cells and CHO-mAb2 cells were cultivated in chemically defined,serum-free medium (Boehringer-Ingelheim) supplemented with 400 nM MTX(Sigma-Aldrich, Germany) and 100 nM MTX, respectively, and G418 (Gibco,Life technologies). Cells were subcultivated every 2 or 3 days with aseeding density of 3×10⁵ cells/mL or 2×10⁵ cells/mL.

Cells were transfected via nucleofection one day after subcultivation(4×10⁵ cells/sample) in 96-well Nucleofector kit SG (Lonza) containing 2μM siRNA using the Amaxa 96-well Shuttle Device (Lonza) and program96-DT-133 according to the manufacturer's instructions. Cells were thenseeded with a density of 3×10⁵ cells/mL into a 24-well flat bottom plate(Greiner). One day after transfection the volume of the medium wasdoubled by addition of fresh medium. Two days after transfection totalRNA was extracted using the RNeasy Plus Mini Kit (Qiagen) and mRNAlevels of target genes ATF6B, CERS2 and TBC1D20 were determined by qPCRas described below. Supernatants were collected at day 1-4 posttransfection and stored at −20° C. until antibody measurement by ELISA.Negative control cells were transfected with water instead of siRNA(mock control) or with a non-targeting negative control pool (NT siRNA)having the following sequences: UGGUUUACAUGUCGACUAA,UGGUUUACAUGUUGUGUGA, UGGUUUACAUGUUUUCUGA and, UGGUUUACAUGUUUUCCUA (SEQID NOs: 38-41, respectively) (Dharmacon, #D-001810-10).

ATF6B, CERS2, TBC1D20, GRP78, CHOP and Herpud1 RNA ExpressionMeasurement by qPCR Analysis

Total RNA of 2×10⁵ to 2×10⁶ cells was extracted using the RNeasy PlusMini Kit (Qiagen). cDNA was generated with 100 ng RNA using theQuantitect Reverse Transcription Kit (Qiagen) according to themanufacturer's instructions. qPCR was performed with the DyNAmoColorFlash SYBR Green qPCR Kit (Thermo Scientific) in a white 96-wellPCR plate (Biorad) using a Cfx96 device (Biorad). Beta actin was used asreference gene. Calculation was done with the single threshold methodand ΔΔCq values were calculated (Bio-rad CFX manager software 2.1).

TABLE 4 Primers used in qPCR analysis Forward primer Reverse primerTarget (5′-3′) (5′-3′) Atf6b GAGCAGGATGTCCCG AGCTCAGGGAGGAGG TTTGA AAGAG(SEQ ID NO: 42) (SEQ ID NO: 43) Tbc1D20 CCCTGAACAGTGATC ATCCTTCCTTGACACCCACC AGGCG (SEQ ID NO: 44) (SEQ ID NO: 45) CerS2 CCCATACAGAGCATCGGCAAACCAGGAGAA GTCCC GCTGA (SEQ ID NO: 46) (SEQ ID NO: 47) CHOPGACCCTGTTTCTTTC GGACTGGGTTCTGCT CCTTCAG TTCAGG (SEQ ID NO: 48)(SEQ ID NO: 49) GRP78 ACCACCTATTCCTGC AGACCGTGTTCTCGG GTTGG GATTG(SEQ ID NO: 50) (SEQ ID NO: 51) Herpud1 GAAGAGTCCCAACCA ATGTCGCTTTTCCTGGCGTC CTTTGG (SEQ ID NO: 52) (SEQ ID NO: 53)

Determination of Recombinant Antibody Concentration

To assess recombinant antibody production in transfected cells,supernatants were collected from cell cultures at the given time points.The product concentration was then analyzed by enzyme linkedimmunosorbent assay (ELISA). First, high binding 96-well microplates(Greiner) were coated with an antibody against the human IgG Fc fragment(Jackson Immuno Research Laboratories) in a 1:480 dilution at 4° C.overnight. After washing three times with 0.15% Tween 20 in PBS (pH 7.4)the plates were incubated with blocking buffer (1% BSA in PBS, pH 7.4)for one hour at room temperature, followed by three washing steps.Supernatants were diluted to an appropriate concentration in the rangeof the standard curve (1-50 ng/μL or 1-100 ng/μl) in dilution buffer(0.5% BSA, 0.01% Tween 80 in PBS, pH 7.4). As a standard, mAb1 antibodywas used in a serial dilution from 0-100 ng/A. Diluted samples andstandards were incubated for 1.5 hours at room temperature. Afterrepeated washing, the samples were incubated with a HRP-conjugatedantibody against the human kappa light chain (Sigma) in a 1:5000dilution for one hour at room temperature, followed by three washingsteps. The substrate p-nitrophenylphosphate (Sigma Aldrich) was freshlyprepared in a concentration of 1 mg/mL with 0.1 M glycine, 1 mM ZnCl₂and 1 mM MgCl2 (pH 10.4) and incubated for 20 minutes in the dark. Thereaction was stopped by addition of 3 M NaOH and absorption at 405 nmand at a reference wavelength of 492 nm was measured using the InfiniteM200 Pro multimode reader (Tecan).

Stable Overexpression of microRNAs miR-1287 and miR-1978

The BLOCK-iT™ Pol II miR RNAi expression vector kit(pcDNA6.2-GW/emGFP-miRNA expression system kit) was used for stablyexpressing miRNAs. DNA oligonucleotides encoding two copies of aspecific microRNA were cloned as short hairpins into the mammalianexpression vector pcDNA6.2. For that purpose, DNA oligonucleotidesencoding the respective miRNAs were designed as described in the manual.In brief, the mature miRNA sequence was embedded in a given sequenceincluding an optimized hairpin loop sequence and 3′ and 5′ flankingregions derived from the murine miRNA mir-155 (Lagos-Quintana et al.,2002). The flanking regions were present on the vector and a DNAoligonucleotide was designed, which encodes the miRNA sequence, thementioned loop and the antisense sequence of the respective mature miRNAwith a 2 nucleotide depletion to generate an internal loop in thehairpin stem. Furthermore, overhangs were added for cloning at bothends. Hairpin structure may be analyzed using the online tool mfold (M.Zuker. Mfold web server for nucleic acid folding and hybridizationprediction. Nucleic Acids Res. 31 (13), 3406-3415, 2003). DNA strandswere annealed and ligated into the 3′-UTR of emerald GFP reporterprotein gene as described by the manufacturer. The oligonucleotidesequences used for cloning of miRNAs into the vector backbone were asfollows:

hsa-miR-1287 oligonucleotide forward: (SEQ ID NO: 28)TGCTGTGCTGGATCAGTGGTTCGAGTCGTTTTGGCCACTGACTGACG ACTCGAACCACATCCAGCAhsa-miR-1287 oligonucleotide reverse: (SEQ ID NO: 29)CCTGTGCTGGATGTGGTTCGAGTCGTCAGTCAGTGGCCAAAACGACT CGAACCACTGATCCAGCAChsa-miR-1978 oligonucleotide forward: (SEQ ID NO: 30)TGCTGGGTTTGGTCCTAGCCTTTCTAGTTTTGGCCACTGACTGACTA GAAAGGCTAACCAAACChsa-miR-1978 oligonucleotide reverse: (SEQ ID NO: 31)CCTGGGTTTGGTTAGCCTTTCTAGTCAGTCAGTGGCCAAAACTAGAA AGGCTAGGACCAAACCC

A vector containing more than one miRNA was generated applying thechaining technique. For one miRNA with two copies, two copies ofspecific microRNAs (e. g. hsa-miR1287 or miR-1978 indicated bypcDNA6.2-GW/emGFP-miR1287-miR1287 or pcDNA6.2-GW/emGFP-miR1978-miR1978)were cloned as DNA oligonucleotides encoding said miRNAs as shorthairpins into the mammalian expression vector pcDNA6.2-GW/emGFP-miRNA(BLOCK-iT™ Pol II miR RNAi expression vector kit, K4936-00 from lifetechnologies) as described by the manufacturer. In brief, the miRNAcassette was excised with the enzymes BamHI and XhoI. The vectorcontaining already one miRNA was opened with the enzymes BglII and XhoI.DNA was mixed with orange loading buffer and was separated in a 1%agarose gel prepared with TAE buffer, bands were visualized withethidium bromide and bands of appropriate size were excised from thegel. Size was verified with DNA ladder. DNA was eluted with the gelextraction kit. DNA insert was ligated into the vector using T4 DNAligase according to manufacturer's instructions. Subsequently, competentE. coli were transformed with the DNA and plated on agar platescontaining spectinomycin. Colonies were picked and DNA was extractedwith a DNA purification kit, checked by control digest with BamHI andBglII first, followed by sequencing.

For generation of stable cell pools CHO-mAb1 were transfected withLipofectamine 2000 and Plus reagent (Invitrogen) using an optimizedprotocol with pcDNA6.2-GW/emGFP-miR-1287-1287 orpcDNA6.2-GW/emGFP-mi-1978-1978 as described by the manufacturer andcells were selected with 10 μg/mL blasticidin S (Life Technologies) andenriched for GFP positive cells by FACS (FACS Diva). As a control vectora negative control miRNA expressing vector (pcDNA6.2-GW/emGFP-neg.control miRNA, provided by the kit) expressing GFP was stablytransfected as described. The negative control miRNA has the followingsequence: GAAAUGUACUGCGCGUGGAGAC (SEQ ID NO: 34)

Stable shRNA-Mediated Knockdown of ATF6B, CERS2 and TBC1D20

The BLOCK-iT™ Pol II miR RNAi expression vector kit(pcDNA6.2-GW/emGFP-miRNA expression system kit) was used for stablyexpressing shRNAs. DNA oligonucleotides encoding specific shRNAs werecloned as short hairpins into the mammalian expression vector pcDNA6.2.For that purpose, DNA oligonucleotides encoding the respective shRNAswere designed and cloned into the integrating vector, as described abovefor the cloning of miRNAs. The oligonucleotide sequences used forcloning of shRNAs into the vector backbone were as follows, wherein theunderline indicates the antisense target site and its complementarysequence with a deletion of two nucleotides, respectively, connectedwith a loop sequences:

shTBC1D20#1 oligonucleotide forward: (SEQ ID NO: 16)TGCTGAATCCTTGCTCAACTGTCGAAGTTTTGGCCACTGACTGACTT CGACAGGAGCAAGGATTshTBC1D20#1 oligonucleotide reverse: (SEQ ID NO: 17)CCTGAATCCTTGCTCCTGTCGAAGTCAGTCAGTGGCCAAAACTTCGA CAGTTGAGCAAGGATTCshCERS2#1 oligonucleotide forward: (SEQ ID NO: 18)TGCTGTTAAGTTCACAGGCAGCCATAGTTTTGGCCACTGACTGACTA TGGCTGTGTGAACTTAAshCERS2#1 oligonucleotide reverse: (SEQ ID NO: 19)CCTGTTAAGTTCACACAGCCATAGTCAGTCAGTGGCCAAAACTATGG CTGCCTGTGAACTTAACshCERS2#2 oligonucleotide forward: (SEQ ID NO: 20)TGCTGTGATGTAGAGGTCTGAGGCTTGTTTTGGCCACTGACTGACAA GCCTCACCTCTACATCAshCERS2#2 oligonucleotide reverse: (SEQ ID NO: 21)CCTGTGATGTAGAGGTGAGGCTTGTCAGTCAGTGGCCAAAACAAGCC TCAGACCTCTACATCACshATF6B#1 oligonucleotide forward: (SEQ ID NO: 22)TGCTGTCCATCTTCACACTGAGGACCGTTTTGGCCACTGACTGACGG TCCTCAGTGAAGATGGAshATF6B#1 oligonucleotide reverse: (SEQ ID NO: 23)CCTGTCCATCTTCACTGAGGACCGTCAGTCAGTGGCCAAAACGGTCC TCAGTGTGAAGATGGACshATF6B#2 oligonucleotide forward: (SEQ ID NO: 35)TGCTGTTCACTTCCAGAACCTCCTCTGTTTTGGCCACTGACTGACAG AGGAGGCTGGAAGTGAAshATF6B#2 oligonucleotide reverse: (SEQ ID NO: 36)CCTGTTCACTTCCAGCCTCCTCTGTCAGTCAGTGGCCAAAACAGAGG AGGTTCTGGAAGTGAAC

As a control vector a negative control miRNA (SEQ ID NO: 34) expressingvector (pcDNA6.2-GW/emGFP-neg. control miRNA, provided by the kit)expressing GFP was stably transfected as described.

A vector containing more than one shRNA(pcDNA6.2-GW/emGFP-shTbc1D20-shCerS2 #1 and pcDNA6.2-GW/emGFP-shCerS2#2-shTbc1D20) was generated applying the chaining technique as describedabove.

For generation of stable cell pools CHO-mAb2 cells were transfected oneday after subcultivation via nucleofection with the Cell LineNucleofector Kit V (Lonza) according to the manufacturer's instructions.In brief, 5×10⁶ cells/sample were resuspended in 100 μL Solution V(Lonza) containing 5 μg plasmid DNA and nucleofected in a cuvette usingthe Cell Line Nucleofector Device (Lonza) and program H14. Cells werethen seeded with 5 mL prewarmed chemically defined, serum-free mediumwithout antibiotics into a T25-flask. 72 hours after transfection themedium was changed to chemically defined, serum-free medium containing 1μg/mL blasticidin S (Life Technologies) for selection. 14 days aftertransfection cells were enriched for GFP positive cells by FACS (BD FACSAria III). Efficient knockdown of ATF6B, CERS2 and TBC1D20 was monitoredby qPCR as described before and cells were analyzed by flow cytometry(Miltenyi MacsQuant) for GFP expression after 42 days in culture.

Knockout of ATF6B, CERS2 and TBC1D20

To generate knockout cells depleted of ATF6B, CERS2 or TBC1D20, theCRISPR/Cas9 technology was applied. For gRNA design, target sites in thegenomic loci of interest selected upstream of protospacer adjacentmotifs (PAM) in the first exons present in all transcript variants ofthe respective genes. For each target gene three gRNA sequences weredesigned and cloned into the GeneArt® CRISPR Nuclease Vector with OFPReporter (Life Technologies) containing a Cas9 nuclease and OFPexpression cassette driven by a CMV promoter and a guide RNA (gRNA)cloning cassette. The guide RNA (consisting of a crRNA specific for thetarget site and a trans activating RNA) expression is driven by a U6pollll type promoter. Subsequently, competent E. coli were transformedwith the DNA and plated on agar plates containing Ampicillin. Colonieswere picked and DNA was extracted with a DNA purification kit andintegrity of the plasmid was analyzed by sequencing. For generation ofstable cells, CHO-mAb2 cells were transfected one day aftersubcultivation via nucleofection with the Cell Line Nucleofector Kit V(Lonza) as described before. Cleavage efficiency was detected using theGeneART® Genomic Cleavage Detection Kit (Life Technologies) according tothe manufacturer's instructions. To generate stable cell clones CHO-mAb2cells were transfected with the described gRNA and Cas9 containingvector and a CMV promoter driven puromycin or alternatively fluorescencemarker gene embedded in flanking regions complementary to genomicregions flanking the target site of interest. Efficient target sitecleavage followed by homology directed repair (HDR) based integration ofthe selection marker gene allows for antibiotic selection and FACSsorting, respectively. Cells with knockout in the ATF6B, CERS2 orTBC1D20 gene were thereby selected and cultured in chemically defined,serum-free medium (Boehringer Ingelheim).

Antibody Purification

The antibody was purified from cell-free cell culture supernatant usingRoboColumns (Atoll) filled with MabSelect resin (GE Healthcare) run on apipetting robot. The low pH used for elution was neutralized to pH 5.5with 1 M TRIS to prevent for antibody denaturation. As amino groupsinterfere with the glycosylation analysis, the buffer was exchanged byultrafiltration using 10 kDa MWCO PES Vivaspin 500 filter units(Sartorius) to pure water. The final protein concentration wasdetermined using a NanoDrop 2000c photospectrometer (Thermo Scientific).

Analysis of the Glycosylation Pattern

To elucidate the structure and composition of the Fc-glycosylation ofIgGs produced in the shRNA expressing CHO-mAb2 cell pools described inExamples 10 and 11, the glycans were released from the purified antibodyafter reduction by enzymatic digestion with PNGase F. The composition ofthe Fc-glycosylation of the IgG antibody was analyzed after PNGaseFrelease and fluorescent labelling using microchip-based capillaryelectrophoresis (CE) with the ProfilerPro Glycan Profiling system on aLabChip GXII instrument (PerkinElmer) according to the manufacturer'sprotocol. Electropherograms were analyzed by the LabChip GX software toidentify and quantify the individual sugar structures. The percentage ofthe glycol-forms was calculated from the chromatographic peak areas. Allvalues were normalized to 100% total sugar structures per sample.

EXAMPLES Example 1: Transcriptome Profiling by Next GenerationSequencing (NGS)

CHO cells are commonly used for the production of therapeutic proteins.Genetic engineering approaches have attempted to optimize theproductivity of these cells by expressing specific cDNAs. Naturallyexisting non-coding RNAs regulate cell fate by modulating the expressionof a whole set of target proteins, which may possibly result in asuper-secretory phenotype when over-expressed in CHO producer cells. Toexploit the power of non-coding RNAs and to identify those thatpositively affect secretion of a heterologous therapeutic protein,CHO-mAb1 cells were transfected with a library of human microRNAs. Basedon this genome-wide functional microRNA (miRNA) screen to identifymiRNAs that enhance the antibody productivity of CHO-mAb1 cells, the twomiRNA screen hits miR-1287 (SEQ ID NO: 32) and miR-1978 (SEQ ID NO: 33)with strong effects on antibody production and specific productivitywere chosen for further analysis (FIG. 1). To identify direct miRNAtarget genes responsible for the positive effects on CHO cellproductivity, CHO-mAb1 cells were transiently transfected with each ofthe two miRNAs. Cells were transfected via nucleofection one day aftersubcultivation (4×10⁵ cells/sample) in 96-well Nucleofector Kit SG(Lonza) as described above. Cells were then seeded with a density of3×10⁵ cells/mL into a 24-well plates (Greiner). 12 hours aftertransfection cells were analyzed by transcriptome profiling using nextgeneration sequencing (NGS) as described above.

Genes that were significantly downregulated with a |log 2| of theirexpression fold change>1 (expression more than 2 fold in untransfectedcells) were defined as hits. Candidate miRNA target genes were thenselected for further analysis with reference to existing knowledge aboutrelevant pathways. ATF6B was chosen as target gene of miR-1287 with theassumption that its downregulation triggers the unfolded proteinresponse (UPR). Both, CERS2 and TBC1D20 were chosen as target genes ofmiR-1978 being involved in the regulation of vesicular and non-vesicularprotein secretion, respectively. Their effects on antibody productionand specific productivity were first assessed by siRNA-mediated geneknockdown. Recapitulating the positive effects on specific productivityin the transient approach, long-term depletion of the target genes wasobtained by shRNAs. Names and sequences of the siRNAs and shRNAs arelisted in FIG. 1B.

Example 2: Selected NGS Hits are Validated by qPCR Analysis

To validate the NGS hits ATF6B and CERS2/TBC1D20 as direct target genesof the miRNAs miR-1287 and miR-1978, respectively, their expressionlevel was analyzed by qPCR. CHO-mAb1 cells were transfected with each ofthe two microRNAs as described above. One day after transfection, RNAwas extracted to measure levels of mRNA of ATF6B, CERS2 or TBC1D20 byqPCR analysis (FIGS. 2A and B) as described above. Relative expressionwas calculated by normalizing to the reference gene beta actin.Additionally, changes in the expression level of the genes of interestwere analyzed by qPCR in CHO-mAb1 cells stably overexpressing therespective miRNAs. These cell pools were generated by transfectingCHO-mAb1 cells with a GFP-containing expression vector further encodingmiR-1287 or miR-1978 (pcDNA6.2-GW/emGFP-miR-1287-1287 andpcDNA6.2-GW/emGFP-miR-1978-1978) (FIGS. 2C and D). Both, CHO-mAb1 cellstransiently and stably expressing miR-1287 showed a reduced expressionlevel of ATF6B, indicating a direct targeting by the miRNA. CHO-mAb1cells expressing miR-1978 transiently as well as cell pools with stablemiR-1978 overexpression showed decreased mRNA levels of CERS2 andTBC1D20. Thus, all the three NGS hits were validated as direct targetgenes of the respective miRNAs.

Example 3: Effective siRNA Mediated Knockdown of ATF6B, CERS2 andTBC1D20

Expression of miR-1287 and miR-1978 strongly improved the specificproductivity of a recombinant antibody expressing CHO cell line(CHO-mAb1). To investigate if the depletion of the validated miR-1287target gene ATF6B and the validated miR-1978 targets CERS2 and TBC1D20also positively affects the specific productivity of recombinantantibody producing CHO cells, CHO-mAb1 cells were transfected withsiRNAs specific for the respective target mRNAs. Three independentsiRNAs were used for downregulation of ATF6B and two siRNAs for CERS2and TBC1D20, respectively. One day after transfection RNA was extractedand mRNA levels of ATF6B, CERS2 and TBC1D20 were quantified by qPCRanalysis. Relative expression was calculated by normalizing to thereference gene beta actin (FIG. 3). CHO-mAb1 cells transientlytransfected with siRNAs specific for ATF6B had strongly reduced levelsof ATF6B mRNA compared to untransfected control cells (and compared tocells transfected with non-targeting control siRNA in a separateexperiment, data not shown). The knockdown efficiency was similar forall three independent siRNAs. Transient transfection of CHO-mAb1 cellswith siRNA specific for CERS2 and siRNA specific for TBC1D20 incombination resulted in reduced expression of CERS2 and TBC1D20 incomparison to untransfected control cells (and compared to cellstransfected with non-targeting control siRNA in a separate experiment,data not shown). Thus, all the siRNAs for ATF6B and for CERS2 as well asfor TBC1D20 induced an effective knockdown of the respective targetgenes.

Example 4: siRNA-Mediated Knockdown of ATF6B Increases the SpecificProductivity of CHO-mAb1 Cells

To investigate if the knockdown of ATf6B improves the specificproductivity of CHO-mAb1 cells stably expressing the mAb1 antibody cellswere transfected with three independent siRNAs specific for ATF6B(siAtf6b #1 (SEQ ID NO: 9), siAtf6b #2 (SEQ ID NO: 10) and siAtf6b #3(SEQ ID NO: 11)). The cells were cultivated for four days in a totalvolume of 1 mL (24 well format) with triplicate samples. Antibodyconcentrations in the supernatant of the transfected cells weredetermined on day 3 and 4 post transfection by ELISA as described above.In addition, cell density and viability were determined each day asdescribed above, enabling the calculation of the specific productivity.Knockdown of ATF6B resulted in an improved specific productivity at day4 (FIG. 4), compared to untransfected control cells (and compared tocells transfected with non-targeting control siRNA in a separateexperiment, data not shown).

Example 5: Combined siRNA-Mediated Knockdown of CERS2 and TBC1D20Increases the Specific Productivity of CHO-mAb1 Cells Up to 1.5-Fold

To explore whether the knockdown of the two miR-1978 targets CERS2 andTBC1D20 has a positive impact on the specific productivity of CHO-mAb1cells, these cells were first transfected with specific siRNAs foreither CERS2 or TBC1D20 and in a second experiment cells weretransfected with both siRNAs together. The experiment was conducted asdescribed in Example 4. Determination of the specific productivity atday 3 and 4 revealed that a single knockdown of either CERS2 or TBC1D20only slightly improved the specific productivity whereas a combinedknockdown of the two target genes increased specific productivity up to1.5 fold (FIG. 5).

Example 6: Three Independent siRNAs for ATF6B Improve SpecificProductivity of CHO-mAb2 Cells

To explore whether the increased specific productivity was specific tomAb1-producing CHO cells or could equally be seen in anotherIgG-producing CHO cell line, CHO-mAb2 cells were transiently transfectedwith each of the three independent siRNAs specific for ATF6B, siAtf6b #1(SEQ ID NO: 9), siAtf6b #2 (SEQ ID NO: 10), siAtf6b #3 (SEQ ID NO: 11).The experiment was conducted as described in Example 4. Remarkably, allthe three siRNAs increased the specific productivity at day 3 and 4 posttransfection 1.2-1.6 fold (FIG. 6).

Example 7: Combined Knockdown of CERS2 and TBC1D20 Improves SpecificProductivity of CHO-mAb2 Cells

Furthermore, we were interested if the combined knockdown of CERS2 andTBC1D20 also leads to an increased specific productivity of CHO-mAb2cells. The experiment was conducted as described in Example 4 usingsiRNA specific for TBC1D20, siTbc1D20 #1 (SEQ ID NO:7) and siRNAspecific for CERS2, siCerS2 #1 (SEQ ID NO: 8). Similar to CHO cellsstably expressing the mAb1 antibody, specific productivity was increasedup to 1.5 fold compared to mock transfected control cells (FIG. 7),providing evidence that this improvement by transient downregulation ofCERS2 and TBC1D20 is independent of the cell clone, the medium and theantibody produced.

A similar increase in specific productivity was also observed forCHO-DG44 cells stably expressing human serum albumin (data not shown).

Example 8: Analysis of shRNA Expression in CHO-mAb2 Cells by FlowCytometry

CHO-mAb2 cells were stably transfected with a plasmid encoding a GFPcassette plus a shRNA sequence comprising a nucleotide sequencespecifically targeting ATF6B (shAtf6b #1; SEQ ID NO: 15) or acombination of two individual shRNA sequences comprising sequencesspecifically targeting CERS2 (shCerS2 #1; SEQ ID NO: 13 and shCerS2 #2;SEQ ID NO: 14) and TBC1D20 (shTbc1D20 #1; SEQ ID NO: 12)(pcDNA6.2-GW/emGFP-shAtf6b #1, pcDNA6.2-GW/emGFP-shTbc1D20 #1-shCerS2 #1and pcDNA6.2-GW/emGFP-shCerS2 #2-shTbc1D20 #1). After selection withblasticidin S (the blasticidin resistance gene is encoded by thepcDNA6.2-GW/emGFP vector) cells were sorted based on their GFPfluorescence. Control cells were untransfected parental cells. Tovalidate the expression of stably transfected shRNAs, cells wereanalyzed by flow cytometry for GFP expression, which correlates withshRNA expression, and GFP positive populations were detected for atleast 42 days (FIG. 8). This shows that CHO cells are able to stablyoverexpress shRNAs for at least 6 weeks.

Example 9: Analysis of shRNA Expression in Stably TransfectedIgG-Producing CHO Cells by Quantitative PCR

CHO-mAb2 cells were stably transfected with a plasmid encoding a GFPcassette plus a shRNA sequence comprising a nucleotide sequencespecifically targeting ATF6B or a combination of two individual shRNAsequences comprising sequences specifically targeting CERS2 and TBC1D20(pcDNA6.2-GW/emGFP-shAtf6b #1, pcDNA6.2-GW/emGFP-shTbc1D20 #1-shCerS2 #1or pcDNA6.2-GW/emGFP-shCerS2 #2-shTbc1D20 #1), as described above. Afterselection with blasticidin S (the resistance gene is encoded on thepcDNA6.2-GW/emGFP vector) cells were sorted based on their GFPfluorescence. Cell pools stably expressing the control vector(pcDNA6.2-GW/emGFP-neg. control) and untransfected parental cells servedas negative controls. To validate the expression of stably transfectedshRNAs, we isolated RNA from all cells and performed qPCR analysis ofthe ATF6B, CERS2 and TBC1D20 mRNA level, respectively, as describedabove. Compared to control vector transfected cells and parental cellsthe cells transfected with shRNA-encoding plasmids had reduced levels ofmRNA of ATF6B or reduced levels of mRNA of both CERS2 and TBC1D20 (FIG.9). This demonstrates that stable genomic integration of plasmid-encodedshRNAs leads to decreased levels of the respective target gene in CHOcells.

Example 10: Fed-Batch Cultivation of CHO-mAb2 Cells Stably Expressing ashRNA for ATF6B

CHO-mAb2 cells were stably transfected with expression vectorscontaining a shRNA sequence comprising a nucleotide sequencespecifically targeting ATF6B (pcDNA6.2-GW/emGFP-shAtf6b #1) and cellswere sorted based on their GFP expression, as described above. One poolof cells expressing a shRNA sequence targeting ATF6B and two independentpools of cells transfected with a negative control vector were usedduring fed-batch cultivation. Cell density, viability and productformation (μg/ml) was determined on days 3-6 by cell counting withtrypan blue exclusion and ELISA analysis, respectively, as describedabove. Specific productivity was calculated. Interestingly, cellsexpressing a shRNA specific for ATF6B showed improved antibody titer,improved specific productivity and an increased viable cell densitycompared to the cells transfected with a negative control vector (FIG.10). Further, cell viability was unaltered (data not shown). This provesthat stable pools of CHO cells depleted of ATF6B have an increasedproduction capacity during fed-batch cultivation without compromisingviable cell density and viability.

Example 11: Fed-Batch Cultivation of CHO-mAb2 Cells Stably Expressing aCombination of shRNAs Specific for CERS2 and TBC1D20

CHO-mAb2 cells were stably transfected with expression vectorscontaining shRNA sequences comprising sequences specifically targetingCERS2 and TBC1D20 (pcDNA6.2-GW/emGFP-shTbc1D20 #1-shCerS2 #1 orpcDNA6.2-GW/emGFP-shCerS2 #2-shTbc1D20 #1) and cells were sorted basedon their GFP expression, as described above. Two independentcombinations of shRNAs targeting the CERS2 and TBC1D20 mRNAs and twoindependent pools of cells transfected with a negative control vectorwere used during fed-batch cultivation in two independent experiments.Cell density, viability and product formation was determined on days3-11 by cell counting with trypan blue exclusion and ELISA analysis,respectively, as described above. Specific productivity was calculated.Both cell pools expressing shRNAs against CERS2 and TBC1D20 showedimproved antibody expression and specific productivity compared to thecell pools transfected with the negative control vector (FIG. 11). Thisproves that stable pools of CHO cells depleted of both CERS2 and TBC1D20have an increased titer and specific productivity capacity.

Example 12: Analysis of Antibody Glycosylation and Antibody AggregateFormation

To analyse product quality we first analysed the structure andcomposition of the Fc-glycosylation of IgGs produced in the shRNAexpressing CHO-mAb2 cell pools described in Examples 10 and 11 above.The glycans were released from the purified antibody after reduction byenzymatic digestion with PNGase F. The composition of theFc-glycosylation of the IgG antibody was analyzed after PNGaseF releaseand fluorescent labelling using microchip-based capillaryelectrophoresis (CE) with the ProfilerPro Glycan Profiling system on aLabChip GXII instrument (PerkinElmer) according to the manufacturer'sprotocol. Electropherograms were analyzed by the LabChip GX software toidentify and quantify the individual sugar structures. The percentage ofthe glycol-forms was calculated from the chromatographic peak areas. Allvalues were normalized to 100% total sugar structures per sample. Theresults in FIG. 12 show that the depletion of ATF6B (FIG. 12A), CERS2and TBC1D20 (FIG. 12B) did not affect glycosylation of the mAb2antibody. In addition to antibodies purified from cells expressingshAtf6b #1, no change in antibody glycosylation was observed inantibodies purified from cells expressing shAtf6b #2 (data not shown).

To further prove that product quality was maintained, the formation ofaggregates was analyzed by HPLC at the end of the fed-batch run.CHO-mAb2 cells, stably expressing shAtf6b #1 or shAtf6b #2(pcDNA6.2-GW/emGFP-shAtf6b #1 or pcDNA6.2-GW/emGFP-shAtf6b #2) or anegative control sequence were cultivated under fed-batch conditions for7 days. The secreted antibody was purified from the supernatants andanalyzed by HPLC. FIG. 12C shows that the antibody preparations from thedifferent cells (shAtf6b #1 or shAtf6b #2 expressing cells as well asthe negative controls) were very similar, with a proportion ofaggregates of less than 1.5%.

A second pool of cells, stably expressing shTbc1D20-shCerS2 #1(pcDNA6.2-GW/emGFP-shTbc1D20 #1-shCerS2 #1) and shCerS #2-shTbc1D20(pcDNA6.2-GW/emGFP-shCerS2 #2-shTbc1D20 #1) or a negative controlsequence were cultivated under fed-batch conditions for 11 days. Thesecreted antibody was purified from the supernatant and analyzed byHPLC. FIG. 12D shows that the antibody preparations from the differentcells (shTbc1D20-shCerS2 #1 or shCerS #2-shTbc1D20 expressing cells aswell as the negative controls) were very similar, with a proportion ofaggregates of less than 1.5%.

Example 13: Expression of Glucose Regulated Protein 78 (GRP78/BiP), CHOPand the ER-Associated Degradation (ERAD) Component HERPUD1 is Increasedin ATF6B Knockdown

As ATF6 acts as a transcription factor during ER stress, we exploredwhether ATF6B knockdown resulted in any changes of UPR-related genes.The mRNA level of three bona fide UPR markers—glucose regulated protein78 (GRP78), homocysteine inducible ER protein with ubiquitin-like domain1 (Herpud1) and CCAAT-enhancer-binding protein homologous protein (CHOPor DDIT3, NCBI Reference Sequence XM_007648092.1) were analyzed—aftertreatment with the ER stress inducing reagent Tunicamyin™.

Stably transfected cells were produced as described in Example 8. Inbrief CHO-mAb2 cells were stably transfected with a plasmid encoding aGFP cassette plus a shRNA sequence comprising a nucleotide sequencespecifically targeting ATF6B (pcDNA6.2-GW/emGFP-shAtf6b #1 orpcDNA6.2-GW/emGFP-shAtf6b #2) encoding shAtf6b #1 comprising thesequence of SEQ ID NO: 15 (UCCAUCUUCACACUGAGGACC) and shAtf6b #2comprising the sequence of SEQ ID NO: 37 (UUCACUUCCAGAACCUCCUCU). Cellpools stably expressing the control vector (pcDNA6.2-GW/emGFP-neg.control) served as negative controls. To validate the expression ofstably transfected shRNAs, RNA was isolated and qPCR analysis of theATF6B mRNA level was performed, as described above. Cells transfectedwith shRNA-encoding plasmids had reduced levels of ATF6B mRNA comparedto negative control (FIG. 13A). This again demonstrates that stablegenomic integration of plasmid-encoded shRNAs targeting ATF6B leads todecreased expression of the respective target gene in CHO cells.

To investigate whether expression of downstream UPR-related genes areaffected CHO-mAb2 cells expressing negative control sequences or shRNAsspecific for ATF6B were treated with or without tunicamycin™. The mRNAlevels of GRP78, Herpud1 and CHOP in untreated cells and TM treatedcells were quantified by qPCR (FIG. 13B). Expression of both GRP78 andHerpud1 was significantly enhanced in cells stably transfected with theshRNAs specific for ATF6B compared to cell pools expressing the negativecontrol construct (black bars), whereas CHOP expression was onlyslightly enhanced. These results indicate that the knockdown of thetranscription factor ATF6B triggers the UPR at the transcriptionallevel.

We further analysed whether expression of downstream UPR-related genesare also affected in a fed-batch culture. The CHO-mAb2 cells stablyexpressing ATF6B-specific shRNAs or the negative control construct werecultivated in shake flasks with daily feeding. On day 5 mRNA levels ofthe UPR-related genes GRP78/BiP, CHOP and Herpud1 were analysed. Inaccordance with the results obtained in seed stock cultures, mRNA levelsof the UPR-related genes GRP78/BiP and Herpud1 were elevated in shATF6Bexpressing cells when compared to control cells (FIG. 13C). Further,shAtf6b #1 and shAtf6b #2 transfected cells showed an increased antibodyconcentration in the supernatant (1.36-fold) and increased viable celldensity at day 7 compared to negative control (data not shown), whilethe viability was unchanged until day 7 and the glycosylation patternwas unaltered (data not shown).

Example 14: TBC1D20 Knockdown Enhances Rab1 Activity and CERS2 KnockdownAlters the Ceramide Composition in Recombinant CHO Cells

TBC1D20 is a GTPase activating protein (GAP) for the small GTPase Rab1.We therefore investigated whether TBC1D20 knockdown in CHO-mAb2 cellsresults in enhanced levels of active Rab1 using a GST pull down assay.

Cloning of p115-GST Expression Construct

To generate a p115-GST fusion protein, p115 (derived from pEGFP-N2-p115as described in Brandon et al. 2006; Mol. Biol. Cell., 17(7): 2996-3008)was subcloned into the vector pGex-6P-1 (GE Healthcare) using KpnI withBamHI and SalI with NotI, respectively. The integrity of the constructwas verified by Sanger sequencing (GATC Biotech AG).

Protein Expression and Extraction

The p115-GST fusion protein was expressed in Escherichia coli, followedby protein extraction. Briefly, transformants were selected in LuriaBroth (LB) medium with 100 μg/mL ampicillin and expression wasstimulated by adding 0.3 mM Isopropyl-β-D-thiogalactopyranosid (IPTG),when the culture reached an OD600 of 0.8-1.0. Bacteria were harvested bycentrifugation and the pellet was frozen on dry ice for 30 minutes. Thepellet was resuspended in lysis buffer (50 mM Tris-HCl at pH 7.5, 1 mMEDTA, 1 mM DTT and complete protease inhibitor (Roche)) and 0.1 μg/mLlysozyme was added. After incubation for 30 minutes on ice with gentleshaking, 5 mM MgCl2 and 20 ng/mL DNAse were added for further 30 minuteson ice, followed by centrifugation at 3000 g for 30 minutes. The proteinextraction supernatant was then incubated with 1 mL 50% glutathionebeads (GE Healthcare) (1 h, 4° C.). Beads were pelleted (5 min at 500 g,4° C.), washed three times with 4 ml washing buffer (lysis buffer plus100 mM NaCl), resuspended in 4 mL resuspension buffer (washing bufferwithout EDTA), pelleted (5 min at 500 g, 4° C.), washed once more andfinally resuspended in 0.5 mL of resuspension buffer resulting in 1 mLof a 50% slurry of GST-p115-beads.

GST Pull Down Assay

CHO-IgG cells were transfected with siTbc1D20, siCerS2 and siTbc1D20 incombination or NT control via nucleofection. 72 hours aftertransfection, 1×10⁶ cells were lysed in 1 mL homogenization buffer (10mM HEPES pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1% Triton-X-100, 1 mM DTT, 5%glycerol, phosphatase inhibitor cocktail and complete protease inhibitorcocktail (Roche)) and incubated for 10 minutes on ice. Each lysate wasmixed with 25 μL of 50% GST-p115-beads and incubated for 1 hour at 4° C.with gentle shaking. Beads were pelleted and washed three times withhomogenization buffer. Beads were resuspended in 20 μL homogenizationbuffer with 10 μL gel loading buffer, incubated at 95° C. for 5 minutes,vortexed briefly and pelleted. Proteins were separated on a 12%acrylamide gel, transferred to nitrocellulose membranes and detectedwith an anti-Rab1 antibody (antibodies—online.com). The fusion proteinwas detected with an anti-GST antibody (GE Healthcare). Aliquots of thecell lysates (input) were also analyzed by immunoblotting using ananti-actin antibody (Sigma-Aldrich) to confirm that equal proteinamounts had been subjected to the pulldown assays

Three days post transfection active Rab1 was extracted from cellhomogenates using the pull-down assay with the p115-GST fusion protein,making use of its binding affinity to the Rab1 effector protein p115fused to GST. CHO-mAb2 cells transfected with siTbc1D20 alone or incombination with siCerS2 showed enhanced amounts of active Rab1 (FIG. 14A). Quantification using Image J software confirmed that Rab1 activitywas increased 2-fold in cells transfected with siTbc1D20 alone or incombination with siCerS2 (FIG. 14B). Knockdown control (qPCR) at day 2post transfection revealed a reduction of Tbc1D20 mRNA by 50% in cellstransfected with siTbc1D20 alone or in combination with siCerS2 (FIG.14C). These findings confirm that the knockdown of the Rab1 GAP TBC1D20amplified Rab1 activity in CHO-mAb2 cells.

We further analysed ceramide composition in cells transfected withsiCerS2 alone or in combination with siTbc1D20. Ceramide synthesis atthe ER and its subsequent transport to the Golgi complex are essentialsteps in the secretory pathway. CERS2 is one of the six ceramidesynthase isoforms expressed in mammalian cells. Each isoform uses arestricted subset of fatty acyl-CoAs as substrates for the ceramidesynthesis. CERS2 preferentially generates very long chain ceramides,particularly C22 and C24-ceramides. Because of its crucial role in thesecretory pathway, it is conceivable that an altered ceramidecomposition of ER membranes could affect sorting and trafficking ofcargo proteins.

To investigate whether the ceramide composition is altered in CERS2knockdown cells, CHO-mAb2 cells were transfected with siCerS2 #1 (SEQ IDNO: 8) alone, in combination with Tbc1D20 #1 (SEQ ID NO: 7) or with anon-targeting siRNA pool (siRNA NT-control, SEQ ID NOs: 38 to 41) as acontrol and analysed using a fluorescent ceramide synthase activityassay.

Fluorescent Ceramide Synthase Activity Assay

At day 3 post nucleofection, 1.5×10⁶ cells were pelleted, resuspended in1 mL lysis buffer (20 mM HEPES at pH 7.4, 25 mMKCl, 2 mM MgCl2, 250 mMSucrose and Complete protease inhibitor cocktail (Roche)) andmechanically lysed using a 26G needle (Terumo). Protein concentrationwas determined using the bicinchoninic acid (BCA) assay (BioRad). Tomeasure ceramide synthase activity, a fluorescent assay was conducted asdescribed by Kim et al (Kim et al., 2012) with minor modifications.Briefly, 50 μg homogenate protein was incubated in reaction buffer (20mM Hepes, pH 7.4, 25 mM KCl, 2 mM MgCl2, 0.5 mM DTT, 0.1% (w/v) fattyacid-free BSA, 10 μM NBD-sphinganine, 50 μM fatty acid-CoA and 250 mMSucrose) with shaking at 35° C. for 30 min (for C16-ceramidequantification) and 120 min (for C24-ceramide quantification). Thereactions were stopped with 250 μL chloroform/methanol (2:1), vortexed,centrifuged and the lower phase was transferred to a 5 mL glass tube.The upper aqueous phase was reextracted as described and both organicphases were combined and dried under a steam of nitrogen, followed byresuspension in 100 μL methanol. 2×2 μL of each reaction were appliedonto aluminum-backed Silica Gel 60 TLC plates and separated bychromatography (chloroform/methanol/water, 8:1:0.1 v/v/v). Fluorescenceof the products was detected using a Typhoon Trio+Scanner (GEHealthcare). The products C16-dihydroceramide and C24-dihydroceramidewere separated by thin layer chromatography and detected using afluorescence scanner (FIG. 14D). Band intensity of C16- andC24-dihydroceramides was quantified using Image J software (FIG. 14E).

In comparison to the control, the knockdown of CERS2 and the combinedknockdown of CERS2 and TBC1D20 resulted in a decreased level ofC24-ceramide and an increased amount of C16-ceramide (FIGS. 14D and E).Quantification revealed that the fraction of C24-ceramide was reduced onaverage by 67.6% after knockdown of CERS2 and 68.8% after combinedknockdown of CERS2 and TBC1D20. C16-ceramide was increased by 73.0% incells transfected with siCerS2 and 19.1% in cells transfected withsiCerS2 and siTbc1D20. Since the reduction of C24-ceramide wasapparently stronger than the enhancement of C16-ceramide, it is possiblethat further LC ceramides (for example C14- or C18-ceramide) areadditionally affected. The efficiency of siRNA-mediated knockdown wasconfirmed by qPCR as described above (FIG. 14F). Thus, the knockdown ofCERS2 results in an altered ceramide composition with an increased ratioof C16-ceramide to C24-ceramide.

Example 15: Stable Knockdown of CERS2 Alters the Ceramide Composition inRecombinant CHO Cells

CHO-mAb2 cells were stably transfected with expression vectorscontaining shRNA sequences comprising sequences specifically targetingCERS2 and TBC1D20 (pcDNA6.2-GW/emGFP-shTbc1D20 #1-shCerS2 #1 orpcDNA6.2-GW/emGFP-shCerS2 #2-shTbc1D20 #1) as described in Example 11.Cells were lysed one day after subcultivation and analysed for ceramidecomposition using a fluorescent ceramide synthase activity assay asdescribed in Example 14.

The double knockdown of CERS2 and TBC1D20 by both independent shRNAcombinations resulted in a strong decrease of C24-ceramide compared tocontrol cells (shTbc1D20 #1-shCerS2 #1: 51.7%, shCerS2 #2-shTbc1D20 #152.1%). At the same time, the amount of C16-ceramide increaseddramatically by 97.7% (shTbc1D20 #1-shCerS2 #1) and 74.5% (shCerS2#2-shTbc1D20 #1), confirming the results obtained in the transientexperiment (FIGS. 14D and E).

Example 16: CRISPR/Cas9-Mediated Knockout of ATF6B in CHO-mAb2 Cells

So far, we could show that transient and stable knockdown of ATF6B inCHO-mAb2 cells enhanced antibody secretion in comparison tonon-transfected cells or cells transfected with a negative controlplasmid. To investigate whether the total depletion of ATF6B proteincould further improve productivity, knockout cells are generated bymutating the ATF6B gene to attain a premature stop of transcription andthus prevent ATF6B protein formation. Applying the CRISPR/Cas9 system,three independent gRNA sequences targeting the ATF6B gene to generateCHO cell clones stably depleted of ATF6B are used. Target sites areselected by identifying protospacer adjacent motifs (PAM) in the firsttwo exons present in both ATF6B transcript variants. Three guide RNAsare designed that each have 20 complementary nucleotides to the sequenceupstream of the PAM sites. Each gRNA is cloned into the GeneArt® CRISPRNuclease Vector with OFP Reporter (Life Technologies). The vectorcontains a CMV promoter driven expression cassette for a Caspase 9 type2 nuclease and an OFP gene and a further expression cassette for theguide RNA that is driven by a U6 pollll type promoter. The vector isamplified in competent E. coli and purified as described before.CHO-mAb2 cells are transfected one day after subcultivation vianucleofection with the Cell Line Nucleofector Kit V (Lonza) as describedbefore. Cleavage efficiency is detected using the GeneART® GenomicCleavage Detection Kit (Life Technologies) according to themanufacturer's instructions. To generate stable cell clones CHO-mAb2cells are transfected with the described gRNA and Cas9 containing vectorand, additionally, a single-stranded CMV promoter driven puromycin oralternatively a fluorescence marker gene. This selection marker gene isembedded in flanking regions complementary to genomic regions flankingthe target site of interest. Only cells in which the genomic ATF6Btarget site is cleaved by the Cas9 nuclease followed by integration ofthe selection marker gene by homology directed repair (HDR) bear theresistance or fluorescence marker, respectively. Single cell clones aregenerated by FACS sorting for OFP positive cells and subsequentcultivation in medium (Boehringer Ingelheim). Efficient depletion ofATF6B protein is controlled, e.g., by PCR or by Western blotting using aspecific anti-ATF6B antibody and efficient mutation in the ATF6B gene isanalyzed by sequencing the genomic loci of interest.

Example 17: CRISPR/Cas9-Mediated Knockout of CERS2 and TBC1D20 inCombination in CHO-mAb2 Cells

We could show before that transient and stable knockdown of CERS2 andTBC1D20 in CHO-mAb2 cells enhanced antibody secretion in comparison tonon-transfected cells or cells transfected with a negative controlplasmid. To investigate whether the total depletion of both CERS2 andTBC1D20 proteins could further improve productivity, knockout cell linesare generated by mutating the CERS2 and TBC1D20 genes to attain apremature stop of transcription and thus prevent CERS2 and TBC1D20protein formation. Applying the CRISPR/Cas9 technology three independentgRNAs targeting CERS2 and three independent gRNAs targeting TBC1D20 areused to mutate the respective genes. Target sites are selected byidentifying protospacer adjacent motifs (PAM) in the first two exonspresent in all six CERS2 transcript variants or two TBC1D20 transcriptvariants, respectively. Three guide RNAs are designed for each gene thathave 20 complementary nucleotides to the sequence upstream of the PAMsites. Each gRNA is cloned into the GeneArt® CRISPR Nuclease Vector withOFP Reporter (Life Technologies) as described above. CHO-mAb2 cells aretransfected as described before and cleavage efficiency is detectedusing the GeneART® Genomic Cleavage Detection Kit (Life Technologies)according to the manufacturer's instructions. To generate stable cellclones CHO-mAb2 cells are transfected with the described gRNA and Cas9containing vector and, additionally, a single-stranded CMV promoterdriven puromycin or alternatively a fluorescence marker gene. Thisselection marker gene is embedded in flanking regions complementary togenomic regions flanking the target site of interest. Only cells inwhich the genomic CERS2 or TBC1D20 target site is cleaved by the Cas9nuclease followed by integration of the selection marker gene byhomology directed repair (HDR) bear the resistance or fluorescencemarker, respectively. Single cell clones are generated by FACS sortingfor OFP positive cells and subsequent cultivation in medium (BoehringerIngelheim). Efficient depletion of CERS2 or TBC1D20 protein iscontrolled, e.g., by PCR or by Western blotting using a specificanti-CERS2 antibody or anti-TBC1D20 antibody, respectively, andefficient mutation in the genome is analyzed by sequencing the genomicloci of interest.

The invention is encompassed by the following items:

1. A mammalian cell having enhanced secretion of a recombinanttherapeutic protein comprising

(a) reduced expression of the host cell proteins TBC1 domain familymember 20 (TBC1D20) and ceramide synthase 2 (CERS2); or(b) reduced expression of the host cell protein activating transcriptionfactor 6 beta (ATF6B), wherein the mammalian cell further comprises oneor more expression cassette(s) encoding a recombinant secretedtherapeutic protein.

2. The mammalian cell of item 1, having reduced ATF6B protein expressionand further comprising one or more expression cassette(s) encoding arecombinant secreted therapeutic protein.

3. The mammalian cell of items 1 or 2, wherein the expression of thehost cell proteins TBC1D20 and CERS2, or of the host cell protein ATF6B,is reduced by at least 30%, at least 40%, at least 50%, at least 75%, or100%, compared to a control mammalian cell.

4. The mammalian cell of any one of items 1 to 3, wherein the secretionof the recombinant therapeutic protein is enhanced by at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 75%, atleast 100% or at least 200%, compared to a control mammalian cell.

5. The mammalian cell of any one of the preceding items, wherein:

(a) the gene encoding the host cell protein comprises a geneticmodification that inhibits expression of said host cell protein, or(b) the mammalian cell comprises a RNA oligonucleotide that inhibitsexpression of the gene encoding said host cell protein byRNA-interference, andwherein the protein expression of TBC1D20 and CERS2 or the proteinexpression of ATF6B in the mammalian cell is reduced compared to thesame mammalian cell not containing said genetic modification(s) or RNAoligonucleotide(s).

6. The mammalian cell of item 5, wherein the RNA-interference ismediated by small hairpin RNA (shRNA) or short interfering RNA (siRNA).

7. The mammalian cell of item 6, wherein the mammalian cell has beentransfected with one or more expression vector(s) comprising anucleotide sequence encoding said siRNA(s) or shRNA(s).

8. The mammalian cell of item 7, wherein the mammalian cell is stablytransfected with one or more expression vector(s) comprising anucleotide sequence encoding said siRNA(s) or shRNA(s).

9. The mammalian cell of any one of items 6 to 8, wherein the siRNA is

(a) siTbc1D20 #1 (SEQ ID NO: 7) or siCerS2 #1 (SEQ ID NO: 8), or acombination thereof; or(b) one or more of siAtf6b #1 (SEQ ID NO: 9), siAtf6b #2 (SEQ ID NO:10), and siAtf6b #3 (SEQ ID NO: 11).

10. The mammalian cell of any one of items 6 to 8, wherein the shRNAcomprises

(a) shTbc1D20 #1 (SEQ ID NO: 12) or one or more of shCerS2 #1 (SEQ IDNO: 13) and shCerS2 #2 (SEQ ID NO: 14), or a combination thereof; or(b) one or more of shAtf6b #1 (SEQ ID NO: 15) and shAtf6b #2 (SEQ ID NO:37), preferably shAtf6b #1 (SEQ ID NO: 15).

11. The mammalian cell of item 5, wherein the genetic modification inthe gene(s) encoding the host cell protein(s) TBC1D20, CERS2 or ATF6B isindependently

(a) a gene deletion; or(b) a mutation in the gene that inhibits expression of the host cellprotein.

12. The mammalian cell of item 11, wherein the mutation is a deletion,addition or substitution.

13. The mammalian cell of item 12, wherein:

(a) the mutation is in the coding region of the gene; and/or(b) the mutation is in the promoter or a regulatory region of the gene.

14. The mammalian cell of any one of the preceding items, wherein:

(a) the host cell protein TBC1D20 has sequence identity of at least 80%to the amino acid sequence of SEQ ID NO: 4 and the host cell proteinCERS2 has sequence identity of at least 80% to the amino acid sequenceof SEQ ID NO: 5; or(b) the host cell protein ATF6B has sequence identity of at least 80% tothe amino acid sequence of SEQ ID NO: 6.

15. The mammalian cell of item 14, wherein

(a) the host cell protein TBC1D20 has the amino acid sequence of SEQ IDNO: 4 and the host cell protein CERS2 has the amino acid sequence of SEQID NO: 5; or(b) the host cell protein ATF6B has the amino acid sequence of SEQ IDNO: 6.

16. A mammalian cell having enhanced secretion of a recombinanttherapeutic protein comprising reduced expression of the host cellproteins TBC1D20 and CERS2.

17. The mammalian cell of item 16, further comprising one or moreexpression cassette(s) encoding a recombinant secreted therapeuticprotein.

18. The mammalian cell of items 17, wherein the secretion of therecombinant therapeutic protein is enhanced by at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 75%, at least100% or at least 200%, compared to a control mammalian cell.

19. The mammalian cell of items 16 to 18, wherein the expression of thehost cell proteins TBC1D20 and CERS2 is reduced by at least 30%, atleast 40%, at least 50%, at least 75%, or 100%, compared to a controlmammalian cell.

20. The mammalian cell of any one of items 16 to 19, wherein:

(a) the gene encoding the host cell protein comprises a geneticmodification that inhibits expression of said host cell protein, or(b) the mammalian cell comprises a RNA oligonucleotide that inhibitsexpression of the gene encoding said host cell protein byRNA-interference, andwherein the protein expression of TBC1D20 and CERS2 in the mammaliancell is reduced compared to the same mammalian cell not containing saidgenetic modifications or RNA oligonucleotides.

21. The mammalian cell of item 20, wherein the RNA-interference ismediated by small hairpin RNA (shRNA) or short interfering RNA (siRNA).

22. The mammalian cell of item 21, wherein the mammalian cell has beentransfected with one or more expression vector(s) comprising anucleotide sequence encoding said siRNA(s) or shRNA(s).

23. The mammalian cell of item 22, wherein the mammalian cell has beenstably transfected with one or more expression vector(s) comprising anucleotide sequence encoding said siRNA(s) or shRNA(s).

24. The mammalian cell of any one of items 21 to 23, wherein the siRNAis siTbc1D20 #1 (SEQ ID NO: 7) or siCerS2 #1 (SEQ ID NO: 8), or acombination thereof.

25. The mammalian cell of any one of items 21 to 23, wherein the shRNAcomprises shTbc1D20 #1 (SEQ ID NO: 12) or one or more of shCerS2 #1 (SEQID NO: 13) and shCerS2 #2 (SEQ ID NO: 14), or a combination thereof.

26. The mammalian cell of item 20, wherein the genetic modification inthe genes encoding the host cell proteins TBC1D20 and CERS2 isindependently

(a) a gene deletion; or(b) a mutation in the gene that inhibits expression of the host cellprotein.

27. The mammalian cell of item 26, wherein the mutation is a deletion,addition or substitution.

28. The mammalian cell of item 27, wherein

(a) the mutation is in the coding region of the gene; and/or(b) the mutation is in the promoter or a regulatory region of the gene.

29. The mammalian cell of any one of items 16 to 28, wherein the hostcell protein TBC1D20 has sequence identity of at least 80% to the aminoacid sequence of SEQ ID NO: 4 and the host cell protein CERS2 hassequence identity of at least 80% to the amino acid sequence of SEQ IDNO: 5.

30. The mammalian cell of any one of items 16 to 29, wherein the hostcell protein TBC1D20 has the amino acid sequence of SEQ ID NO: 4 and thehost cell protein CERS2 has the amino acid sequence of

SEQ ID NO: 5.

31. The mammalian cell of any one of the preceding items, wherein thesaid recombinant secreted therapeutic protein is

(a) an antibody, preferably a monoclonal antibody, a bi-specificantibody or a fragment thereof, or(b) an Fc-fusion protein.

32. The mammalian cell of any one of the preceding items, wherein thecell is a rodent or a human cell.

33. The mammalian cell of item 32, wherein the rodent cell is a hamstercell.

34. The mammalian cell of item 33, wherein the hamster cell is a CHOcell, preferably a CHO-DG44 cell, a CHO-K1 cell or a glutaminesynthetase (GS)-deficient derivative thereof.

35. A method of producing a mammalian cell with enhanced secretion of arecombinant therapeutic protein comprising

(a) reducing expression of the host cell proteins TBC1D20 and CERS2, orof the host cell protein ATF6B in the mammalian cell by introducing(i) a genetic modification into a gene encoding the host cell proteinthat inhibits expression of said host cell protein, or(ii) a RNA oligonucleotide into the mammalian cell that inhibitsexpression of the gene encoding said host cell protein byRNA-interference, and(b) introducing one or more gene(s) encoding a recombinant secretedtherapeutic protein.

36. The method of item 35, further comprising the following steps:

(c) selecting cells with enhanced secretion of the recombinanttherapeutic protein; and(d) optionally culturing the cells obtained in step (c) under conditionswhich allow expression of one or more gene(s) encoding a recombinantsecreted therapeutic protein.

37. The method of items 35 or 36, wherein the protein expression ofTBC1D20 and CERS2 or the protein expression of ATF6B in the mammaliancell is reduced compared to the same mammalian cell not containing saidgenetic modification(s) or RNA oligonucleotide(s).

38. The method of any one of items 35 or 37, comprising reducing TBC1D20and CERS2 protein expression in the mammalian cell.

39. The method of any one of items 35 or 37, comprising reducing ATF6Bprotein expression in the mammalian cell.

40. The method of any one of items 35 to 39, wherein theRNA-interference is mediated by small hairpin RNA (shRNA) or shortinterfering RNA (siRNA).

41. The method of item 40, comprising transfecting the mammalian cellwith one or more expression vector(s) comprising a nucleotide sequenceencoding said siRNA(s) or shRNA(s).

42. The method of item 41, wherein the mammalian cell is stablytransfected with one or more expression vector(s) comprising anucleotide sequence encoding said siRNA(s) or shRNA(s).

43. The method of any one of items 40 to 42, wherein the siRNA is

(a) siTbc1D20 #1 (SEQ ID NO: 7) or siCerS2 #1 (SEQ ID NO: 8), or acombination thereof; or(b) one or more of siAtf6b #1 (SEQ ID NO: 9), siAtf6b #2 (SEQ ID NO:10), and siAtf6b #3 (SEQ ID NO: 11).

44. The method of any one of items 40 to 42, wherein the shRNA comprises

(a) shTbc1D20 #1 (SEQ ID NO: 12) or one or more of shCerS2 #1 (SEQ IDNO: 13) and shCerS2 #2 (SEQ ID NO: 14), or a combination thereof; or(b) one or more of shAtf6b #1 (SEQ ID NO: 15) and shAtf6b #2 (SEQ ID NO:37), preferably shAtf6b #1 (SEQ ID NO: 15).

45. The method of item 35, wherein the genetic modification in thegene(s) encoding the host cell protein(s) TBC1D20 and CERS2, or ATF6B isindependently

(a) a gene deletion; or(b) a mutation in the gene that inhibits expression of the host cellprotein.

46. The method of item 45, wherein the mutation is a deletion, additionor substitution.

47. The method of item 46, wherein:

(a) the mutation is in the coding region of the gene; and/or(b) the mutation is in the promoter or a regulatory region of the gene.

48. The method of any one of items 35 to 47, wherein:

(a) the host cell protein TBC1D20 has sequence identity of at least 80%to the amino acid sequence of SEQ ID NO: 4 and the host cell proteinCERS2 has sequence identity of at least 80% to the amino acid sequenceof SEQ ID NO: 5; or(b) the host cell protein ATF6B has sequence identity of at least 80% tothe amino acid sequence of SEQ ID NO: 6.

49. The method of any one of items 35 to 48, wherein

(a) the host cell protein TBC1D20 has the amino acid sequence of SEQ IDNO: 4 and the host cell protein CERS2 has the amino acid sequence of SEQID NO: 5; or(b) the host cell protein ATF6B has the amino acid sequence of SEQ IDNO: 6.

50. The method of any one of items 35 to 49, wherein the saidrecombinant secreted therapeutic protein is

(a) an antibody, preferably a monoclonal antibody, a bi-specificantibody or a fragment thereof, or(b) an Fc-fusion protein.

51. The method of any one of items 35 to 50, wherein the cell is arodent or a human cell.

52. The method of item 51, wherein the rodent cell is a hamster cell.

53. The method of item 52, wherein the hamster cell is a CHO cell,preferably a CHO-DG44 cell, a CHO-K1 cell or a glutamine synthetase(GS)-deficient derivative thereof.

54. The method of any one of items 35 to 53, wherein step (a) may beperformed before or after step (b).

55. The method of any one of items 35 to 54, wherein the selection stepof step (c) is performed in the presence of a selection agent.

56. The method of any one of items 35 to 55, further comprising anamplification step (b′), comprising amplifying the one or more gene(s)introduced in step (b) together with an amplifiable selectable markergene and culturing the mammalian cell in the presence of an agent whichallows the amplification of the amplifiable selectable marker gene.

57. The method of items 56, wherein the amplifiable selectable markergene encodes the amplifiable selectable markers dihydrofolate reductaseor glutamine synthetase.

58. The method of any one of items 35 to 57, wherein the expression ofthe host cell proteins TBC1D20 and CERS2 or of the host cell proteinATF6B is reduced by at least 30%, at least 40%, at least 50%, at least75%, or 100% compared to a mammalian cell produced by the method of anyone of items 35 to 56 using the same starting material, but omittingstep (a).

59. The method of any one of items 35 to 58, wherein the secretion ofthe recombinant therapeutic protein is enhanced by at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 75%, atleast 100% or at least 200% compared to a mammalian cell produced by themethod of any one of items 35 to 58 using the same starting material,but omitting step (a).

60. A method for the production of a recombinant secreted therapeuticprotein in a mammalian cell comprising

(a) providing the mammalian cell of any one of items 1 to 34 wherein thecell is transfected with a recombinant secreted therapeutic protein orproviding the mammalian cell produced by the method of any one of theitems 35 to 59,(b) culturing the mammalian cell of step (a) in a cell culture medium atconditions allowing production of the recombinant secreted therapeuticprotein,(c) harvesting the recombinant secreted therapeutic protein, andoptionally(d) purifying the recombinant secreted therapeutic protein.

61. Use of the mammalian cell of any one of items 1 to 34 or themammalian cell produced by the method of items 35 to 59 for increasingthe yield of a recombinant secreted therapeutic protein.

62. The use of item 61, wherein the recombinant secreted therapeuticprotein is

(a) an antibody, preferably a monoclonal antibody, a bi-specificantibody or a fragment thereof, or (b) an Fc-fusion protein.

Sequence Table

SEQ ID NO 1: Tbc1D20 Chinese Hamster cDNA

SEQ ID NO 2: CerS2 Chinese Hamster cDNA

SEQ ID NO 3: Atf6b Chinese Hamster cDNA

SEQ ID NO 4: TBC1D20 Chinese Hamster protein

SEQ ID NO 5: CERS2 Chinese Hamster protein

SEQ ID NO 6: ATF6B Chinese Hamster protein

SEQ ID NO 7: siTbc1D20 #1

SEQ ID NO 8: siCerS2 #1

SEQ ID NO 9: siAtf6b #1

SEQ ID NO 10: siAtf6b #2

SEQ ID NO 11: siAtf6b #3

SEQ ID NO 12: shTbc1D20 #1

SEQ ID NO 13: shCerS2 #1

SEQ ID NO 14: shCerS2 #2

SEQ ID NO 15: shAtf6b #1

SEQ ID NO 16: shTbc1D20 #1_oligonucleotide_forward

SEQ ID NO 17: shTbc1D20 #1_oligonucleotide_reverse

SEQ ID NO 18: shCerS2 #1_oligonucleotide_forward

SEQ ID NO 19: shCerS2 #1_oligonucleotide_reverse

SEQ ID NO 20: shCerS2 #2_oligonucleotide_forward

SEQ ID NO 21: shCerS2 #2_oligonucleotide_reverse

SEQ ID NO 22: shAtf6b #1_oligonucleotide_forward

SEQ ID NO 23: shAtf6b #1_oligonucleotide_reverse

SEQ ID NO 24: mAb1 IgG1 heavy chain

SEQ ID NO 25: mAb1 IgG1 light chain

SEQ ID NO 26: mAb2 IgG1 heavy chain

SEQ ID NO 27: mAb2 IgG1 light chain

SEQ ID NO 28: hsa-miR-1287_oligonucleotide_forward

SEQ ID NO 29: hsa-miR-1287_oligonucleotide_reverse

SEQ ID NO 30: hsa-miR-1978_oligonucleotide_forward

SEQ ID NO 31: hsa-miR-1978_oligonucleotide_reverse

SEQ ID NO 32: hsa-miR-1287

SEQ ID NO 33: hsa-miR-1978

SEQ ID NO:34: negative control

SEQ ID NO 35: shAtf6b #2_oligonucleotide_forward

SEQ ID NO 36: shAtf6b #2_oligonucleotide_reverse

SEQ ID NO 37: shAtf6b #2

SEQ ID NO 38: siRNA NT-control #1

SEQ ID NO 39: siRNA NT-control #2

SEQ ID NO 40: siRNA NT-control #3

SEQ ID NO 41: siRNA NT-control #4

SEQ ID NO 42: qPCR primer (Atf6b) forward

SEQ ID NO 43: qPCR primer (Atf6b) reverse

SEQ ID NO 44: qPCR primer (Tbc1D20) forward

SEQ ID NO 45: qPCR primer (Tbc1D20) reverse

SEQ ID NO 46: qPCR primer (CerS2) forward

SEQ ID NO 47: qPCR primer (CerS2) reverse

SEQ ID NO 48: qPCR primer (CHOP) forward

SEQ ID NO 49: qPCR primer (CHOP) reverse

SEQ ID NO 50: qPCR primer (GRP78) forward

SEQ ID NO 51: qPCR primer (GRP78) reverse

SEQ ID NO 52: qPCR primer (Herpud1) forward

SEQ ID NO 53: qPCR primer (Herpud1) reverse

1. A Chinese Hamster Ovary (CHO) cell engineered to have enhancedsecretion of a recombinant therapeutic protein, compared to anon-engineered CHO cell, comprising reduced expression of the host cellprotein activating transcription factor 6 beta (ATF6B), wherein the CHOcell further comprises one or more expression cassette(s) encoding arecombinant secreted therapeutic protein.
 2. (canceled)
 3. Theengineered CHO cell of claim 1, further comprising one or moreexpression cassette(s) encoding a recombinant secreted therapeuticprotein.
 4. The engineered CHO cell of claim 1, wherein: (a) the geneencoding the host cell protein comprises a genetic modification thatinhibits expression of said host cell protein, or (b) the CHO cellcomprises a RNA oligonucleotide that inhibits expression of the geneencoding said host cell protein by RNA-interference, and wherein theprotein expression of ATF6B in the CHO cell is reduced compared to thesame CHO cell not containing said genetic modification(s) or RNAoligonucleotide(s). 5-10. (canceled)
 11. The engineered CHO cell ofclaim 4, wherein the genetic modification in the gene(s) encoding thehost cell protein ATF6B is independently (a) a gene deletion; or (b) amutation in the gene that inhibits expression of the host cell protein,wherein said mutation is in the coding region of the gene and/or thepromoter or regulatory region of the gene.
 12. The engineered CHO cellof claim 1, wherein: the host cell protein ATF6B has sequence identityof at least 80% to the amino acid sequence of SEQ ID NO:
 6. 13. Theengineered CHO cell of claim 1, wherein the said recombinant secretedtherapeutic protein is (a) an antibody, a monoclonal antibody, abi-specific antibody or a fragment thereof, or (b) an Fc-fusion protein.14-15. (canceled)